Electrophoretic system with multi-notch filter and laser excitation source

ABSTRACT

Apparatus and methods for detecting analytes in a sample are provided. The apparatus can include: one or more channels having a detection zone; one or more irradiation sources disposed for irradiating the detection zone with radiation; a detector capable of collecting at least one charge corresponding to an emission beam emitted from the detection zone, the detector having an output; and a time delay integration system coupled to the detector for effecting time delay integration of the at least one charge by accumulating the at least one charge before reading the at least one charge at the output of the detector. Various embodiments provide an apparatus with a multi-notch filter. Various embodiments provide a solid state laser, a micro-wire laser, or an organic light-emitting diode as a irradiation source.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/205,028 filed Jul. 25, 2002, which claims a benefit fromU.S. Provisional Application No. 60/307,682 filed Jul. 25, 2001, andthis application also claims benefit under 35 U.S.C. Section 119(e) fromearlier U.S. Provisional Patent Application No. 60/485,468, filed Jul.8, 2003, and this application also claims benefit under 35 U.S.C.Section 119(e) from earlier U.S. Provisional Patent Application No.60/486,112, filed Jul. 10, 2003, all of which are incorporated herein intheir entireties by reference. U.S. patent application Ser. No.10/805,096, filed Mar. 19, 2004, to Reel et al. is also incorporatedherein in its entirety by reference.

FIELD

The present teachings relate to electrophoretic detection systems andlight filters and light sources for such systems.

BACKGROUND

Well-known examples of biopolymer analysis using DNA sequencing aretaught, for example, in F. Sanger et al., DNA Sequencing with ChainTerminating Inhibitors, 74 Proc. Nat. Acad. Sci. USA 5463 (1977); LloydM. Smith et al., Fluorescence detection in automated DNA sequenceanalysis, 321 Nature 674 (1986); and Lloyd M. Smith, The Future of DNASequencing, 262 Science 530 (1993). These and all other publications andpatents cited herein are incorporated herein in their entireties byreference.

An electrophoretic apparatus and method that includes a cost-effectiveand convenient source of irradiation and that does not compromisesensitivity or resolution would be desirable, especially inmultiple-channel electrophoretic systems used to increase throughput.

SUMMARY

According to various embodiments, an apparatus for detecting components,for example, analytes, in a sample is provided. The apparatus caninclude: one or more channels having a detection zone; one or moreirradiation sources disposed for irradiating the detection zone withradiation; a detector capable of collecting at least one chargecorresponding to an emission beam emitted from the detection zone, thedetector having an output; a time delay integration system coupled tothe detector for effecting time delay integration of the at least onecharge by accumulating the at least one charge before reading the atleast one charge at the output of the detector, and modulating opticsincluding a multi-notch filter disposed along a beam path between theone or more irradiation sources and the detection zone and/or betweenthe detection zone and the detector. Methods of analysis using theapparatus are also provided.

According to various embodiments, an apparatus for detecting components,for example, analytes, in a sample is provided. The apparatus caninclude: one or more channels having a detection zone; one or moreirradiation sources disposed for irradiating the detection zone withradiation, wherein the one or more irradiation sources includes a solidstate laser source, a micro-wire laser source, or an organiclight-emitting diode; a detector capable of collecting at least onecharge corresponding to an emission beam emitted from the detectionzone, the detector having an output; and a time delay integration systemcoupled to the detector for effecting time delay integration of the atleast one charge by accumulating the at least one charge before readingthe at least one charge at the output of the detector. Method ofanalysis using the apparatus are also provided.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory only.The accompanying drawings, which are incorporated in and constitute apart of this application, illustrate several exemplary embodiments and,together with the instant description, serve to explain the principlesof the present teachings.

BRIEF DESCRIPTION OF DRAWINGS

Various embodiments of the present teachings are exemplified in theaccompanying drawings. The teachings are not limited to the embodimentsdepicted, and include equivalent structures and methods as set forth inthe following description and known to those of ordinary skill in theart. In the drawings:

FIG. 1 is a schematic, side-elevational view of an electrophoresisarrangement showing a channel-defining member in cross-section,according to various embodiments;

FIG. 2 is a schematic view of an image produced on a detector array of adetector at a time t using the arrangement of FIG. 1;

FIG. 3 is a view similar to FIG. 2 showing the image at a time t+Δt;

FIG. 4 is a schematic, front-elevational view of an electrophoresisarrangement for the sequential use of multiple-color irradiation sourcesalong with filters on a filter wheel, according to various embodiments;

FIG. 5 a is a schematic, top-plan view of the arrangement of FIG. 4;

FIG. 5 b is a schematic, side-elevational view of the arrangement ofFIG. 4;

FIGS. 6 a through 6 e are respective schematic views of images producedon the detector array of a detector, each image corresponding to lightsignals filtered through a respective filter on the filter wheel of FIG.4;

FIG. 6 f is a schematic representation of an electropherogram showingfluorescence intensity curves for each filter of the filter wheel inFIG. 4 during three signal readings by the detector;

FIG. 6 g is a schematic representation of the intensity curves of FIG. 6f in aligned format for multicomponenting;

FIG. 6 h is a schematic representation of multicomponented intensitycurves for five different kinds of markers that can be used in thesystem of FIG. 4 based on the readings shown in FIG. 6 f;

FIG. 6 i is a graph of excitation efficiency versus wavelength for fourexemplary markers;

FIG. 6 j is a graph of fluorescence intensity versus wavelength for theexemplary markers of FIG. 6 i;

FIG. 7 is a top-plan, partially cross-sectional view, of anelectrophoresis arrangement for the sequential use of multiple colorirradiation sources along with filters on a filter wheel and along withan offset system, according to various embodiments;

FIG. 8 is a schematic view of an image produced on the detector array ofa detector using the arrangement of FIG. 7;

FIG. 9 a is a graph of relative excitation intensity versus wavelengthfor a pair of LEDs used in another embodiment, the LEDs can be ofdifferent colors and can be used to irradiate the detection zonesimultaneously;

FIG. 9 b is a graph showing percent transmission versus wavelength for aconditioning filter used to condition the light from the LEDs of FIG. 9a;

FIG. 9 c is a graph showing percent transmission versus wavelength for abandpass filter used to filter light signals produced by markers excitedby the light from the LEDs of FIG. 9 a;

FIG. 9 d is a graph showing relative emission intensity versuswavelength for the light filtered through the bandpass filter of FIG. 9c;

FIG. 10 is a schematic, top-plan view of an irradiation zone showingthree channels having been selectively masked to present respectivewindows according to another embodiment;

FIG. 11 is a schematic view of a detector array, the detector arrayhaving been separated into respective frames for use with light signalsemitted from the respective windows of the channels in FIG. 10;

FIG. 12 a is a partially cut away view of one of the frames of thedetector array shown in FIG. 11;

FIG. 12 b is a graph showing percent integration per pixel throughoutthe width of the frame as shown in FIG. 12 a;

FIG. 13 a is a schematic of a portion of an electrophoresis arrangementshowing electrostatic sorting of the components, according to variousembodiments;

FIG. 13 b is a schematic of a portion of an electrophoresis arrangementshowing mechanical sorting of the components, according to variousembodiments; and

FIG. 13 c is a schematic of a portion of an electrophoresis arrangementshowing sorting of the components into various channels of amulti-channel microcard, according to various embodiments.

FIG. 14 illustrates an exemplary embodiment of a light source layout,for example, an organic light emitting diode (OLED) layout with varyingcolor OLEDs stacked upon each other.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are intended to provide a further explanation of the variousembodiments of the present teachings.

DESCRIPTION

Exemplary analytes can include nucleic acids, both single and doublestranded, proteins, carbohydrates, viruses, cells, organelles, organicpolymers, particles, labeled media, labeled beads, and the like. Theanalytes can include biomolecules, for example, cells, proteins, DNA,RNA, polynucleotides, polypeptides, polysaccharides, and small moleculeanalytes. According to various embodiments, the analyte can be aselected-sequence polynucleotide, and an analyte-specific reagentincluding a sequence-selective reagent for detecting the polynucleotidecan be associated with the polynucleotide. Polynucleotide analytes canbe detected by any suitable method, for example, polymerase chainreaction, ligase chain reaction, oligonucleotide ligation assay,hybridization assay, antibody assay, affinity assay, orstreptavidin/biotin assay.

The analytes of interest can be detected on the basis of anintrinsically detectable signal, or may be derivatized with a labelwhich confers a desired type of detectability. Various methods forlabeling analytes with detectable moieties are well known in the art,such as excitable reporters, radioactive isotopes, fluorescent dyes,spin labels, chemiluminescent compounds and the like to stimulatedetectable emission indicative of the nature of the analyte. When theanalyte is a polynucleotide, labeling by hybridization with a labeledprobe can also be used. The analyte can be labeled with a dye marker, afluorescing dye, a free-floating dye, a reporter dye, a probe dye, anintercalating dye, a quantum dot, a molecular beacon, a linear probe, ora combination thereof.

As used herein, the term “separation medium” or “separation matrix”refers to a medium in which an electrophoretic separation of samplecomponents can take place. Separation media can include severalcomponents, at least one of which can be a charge-carrying component, orelectrolyte. The charge-carrying component can be part of a buffersystem for maintaining the separation medium at a defined pH. Media forseparating polynucleotides, proteins, or other biomolecules can havedifferent sizes but identical charge-frictional drag ratios in a freesolution, further include a sieving component. Such sieving componentscan include a cross-linked polymer gel, for example, a cross-linkedpolyacrylamide or agarose (Sambrook), or a polymer solution, such as, asolution of polyacrylamide, hydroxyethyl cellulose, and the like(Grossman; Madabhushi).

According to various embodiments, an apparatus is provided that includesa time-delay integration detection system and an optical pathway thatincludes one or more multi-notch filters, for example, one or moreRugate filters. According to various embodiments, an apparatus isprovided that includes a time-delay integration detection system and anexcitation light source that includes one or more of an organiclight-emitting diode, a microwave laser source, and a solid state lasersource.

According to various embodiments, a method for detecting components, forexample, analytes, in a sample using at least one marker is provided.The method can include the steps of: providing one or more channelshaving a detection zone; providing migration medium within the one ormore channels; separating a sample containing analytes and disposed incontact with the migration medium into analyte bands migrating along theone or more channels, wherein each analyte band is detectable by thepresence of the at least one marker; irradiating the detection zoneusing one or more irradiation sources disposed for irradiating thedetection zone with radiation so that the at least one marker isresponsive to the radiation to produce emission beams indicative ofcorresponding analytes; detecting the emission beams on a detector toproduce at least one charge on the detector corresponding to theemission beams; modulating or filtering light between the one or moreirradiation sources and detection zone or between the detection zone andthe detector wherein modulating or filtering the light includesdirecting the light through a multi-notch filter; effecting a time delayintegration of the at least one charge by accumulating the at least onecharge associated with at least one given analyte band during anintegration time of the at least one given analyte before reading the atleast one charge at the output of the detector; and reading theaccumulated charges. For example, the multi-notch filter can be disposedalong the excitation beam path, and a high pass filter can be disposedalong the emission beam path.

According to various embodiments, a method for detecting components, forexample, analytes, in a sample using at least one marker is provided.The method can include the steps of: providing one or more channelshaving a detection zone; providing migration medium within the one ormore channels; separating a sample containing analytes and disposed incontact with the migration medium into analyte bands migrating along theone or more channels, wherein each analyte band is detectable by thepresence of the at least one marker; irradiating the detection zoneusing one or more irradiation sources disposed for irradiating thedetection zone with radiation so that the at least one marker isresponsive to the radiation to produce emission beams indicative ofcorresponding analytes wherein the one or more irradiation sourcesincludes a solid state laser source, a micro-wire laser source, or anorganic light-emitting diode; detecting the emission beams on a detectorto produce at least one charge on the detector corresponding to theemission beams; modulating light between the one or more irradiationsources and the detection zone and/or between the detection zone and thedetector; effecting a time delay integration of the at least one chargeby accumulating the at least one charge associated with at least onegiven analyte band during an integration time of the at least one givenanalyte before reading the at least one charge at the output of thedetector; and reading the accumulated charges.

According to various embodiments, an apparatus or method for detectingcomponents, for example, analytes, in a sample is provided. Theapparatus can include: one or more channels having a detection zone; oneor more irradiation sources disposed for irradiating the detection zonewith radiation to thereby excite markers responsive to the radiationcausing the markers to emit light signals indicative of correspondinganalytes; a detector array disposed for collecting the light signalsemitted from markers in the detection zone excited by the radiation andfor producing charges corresponding to the light signals, where thedetector array can have an output; and a time-delay integration systemcoupled to the detector array for effecting time delay integration ofthe charges on the detector array corresponding to the light signalsassociated with at least one analyte band by accumulating the chargesbefore reading the charges at the output of the detector array, theaccumulation can be effected during an integration time of the at leastone given analyte band moving across the detection zone. Time delayintegration can be effected by moving, relative to one another, thedetector array and light signals from the detection zone; the detectorarray and modulating optics; or the sample to be detected and thedetection system, wherein the detection system includes the detectorarray and one or more irradiation sources.

The apparatus for detecting analytes in a sample can include are-imaging lens disposed between the detection zone and the detectorarray for optically inverting an image produced by the light signalsbefore the image can be collected by the detector array. The apparatuscan include modulating optics for modulating light between theirradiation source and the detector array.

According to various embodiments, the apparatus for detecting analytesin a sample can include a separating system coupled to the one or morechannels for separating a sample containing analytes into analyte bandsmigrating along the one or more channels, wherein each analyte band canbe detected by the presence of a corresponding marker, and wherein thesample containing analytes can be disposed in contact with a migrationmedium and disposed within the one or more channels.

According to various embodiments, the apparatus can include a sortingsystem for separating each detected analyte into a respective collectionof analytes. The sorting system can include a motive force for directingthe analyte to an analyte collector, wherein the collector can be adish, channel, capillary tube, beaker, or other device capable ofretaining the analyte. The motive force can act on the analyte, on thecollector, or both. The motive force can be an electro-kinetic force, amechanical force, an electric field gradient, a vacuum, or a combinationthereof.

According to various embodiments, an apparatus for detecting analytes ina sample can include: means defining one or more channels therein havinga detection zone; means for separating a sample containing analytes anddisposed in contact with a migration medium disposed within the one ormore channels into analyte bands migrating along the one or morechannels, wherein each analyte band can be detectable by the presence ofa marker; means for irradiating the detection zone with radiation, thatcan thereby excite markers responsive to the radiation and that can emitlight signals indicative of corresponding analytes; means for detectingthe light signals by collecting the light signals that can therebyproduce charges corresponding thereto; means for effecting a time delayintegration of the light signals within the detector array by, forexample, accumulating within the detector array the chargescorresponding to light signals associated with at least one givenanalyte band during an integration time of the at least one givenanalyte band moving across the detection zone; and means for reading theaccumulated charges The apparatus can also include means for sorting thedetected analytes, a means for multi-notch filtering the non-coherentradiation, a means for multi-notch filtering emitted light, and/or anexcitation source including one or more of a solid state laser source, amicrowire laser source, and an organic light-emitting diode.

According to various embodiments, an apparatus and method for sortingcomponents, for example, analytes, in a sample is provided. Theapparatus can include: one or more channels having a detection zone; oneor more irradiation sources disposed for irradiating the detection zonewith radiation; a detector array disposed for collecting light signalsemitted from markers in the detection zone excited by the radiation,wherein the detector array can have an output; and a means for directinga detected component into one or more pathways, for example, byelectrokinetic movement or mechanical movement of the detected componentor of the pathway for receiving the detected component. The apparatuscan further include a system coupled to the detector array for effectingtime delay integration of the charges on the detector arraycorresponding to the light signals by accumulating the charges beforereading the charges at the output of the detector array.

According to various embodiments, an apparatus for sorting analytes in asample is provided. The apparatus can include a channel-defining memberdefining one or more channels therein having a detection zone; and aseparating system coupled to the one or more channels for separating asample containing analytes into analyte bands migrating along the one ormore channels, wherein each analyte band can be detected by the presenceof a corresponding marker, and wherein the sample containing analyte canbe disposed in contact with the migration medium and disposed within theone or more channels. The apparatus can include one or more irradiationsources for providing excitation radiation and disposed for irradiatingthe detection zone of the one or more channels to thereby excite markersresponsive to the radiation, causing them to emit light signalsindicative of corresponding analytes; a detector array disposed forcollecting the light signals produced by the markers and for producingcharges corresponding to the light signals, where the detector array canhave an output; modulating optics for modulating light between theirradiation source and the detection zone and/or between the detectionzone and the detector array; and a means of sorting the detectedanalytes into various pathways, for example, by electrokinetic force ormechanical force. According to various embodiments, the apparatus caninclude a time delay integration system for effecting, within thedetector array, an accumulation of charges corresponding to lightsignals associated with at least one given analyte band before readingaccumulated charges at the output of the detector array, theaccumulation can be effected during integration time of the at least onegiven analyte band moving across the detection zone. The apparatus caninclude a re-imaging optical system disposed between the detection zoneand the detector array for optically inverting an image produced by thelight signals before the image can be collected by the detector array.The modulating optics can include one or more multi-notch filters, forexample, one or more Rugate filters.

A method for detecting analytes in a sample can include: providing achannel-defining member defining one or more channels therein having adetection zone; providing migration medium within the one or morechannels; separating a sample containing analytes and disposed incontact with the migration medium into analyte bands migrating along theone or more channels, wherein each analyte band can be detected by thepresence of a marker; irradiating the detection zone using one or moreirradiation sources providing radiation that can thereby excite markersresponsive to the radiation and that can emit light signals indicativeof corresponding analytes; detecting the light signals produced by themarkers by, for example, collecting the light signals on a detectorarray to produce charges on the detector array corresponding to thelight signals; modulating light between the one or more irradiationsources and the detection zone and/or between the detection zone and thedetector array; effecting a time delay integration of the light signalswithin the detector array by, for example, accumulating the chargeswithin the detector array corresponding to light signals associated withat least one given analyte band during an integration time of the atleast one given analyte band moving across the detection zone; readingthe accumulated charges; and sorting a detected analyte by, for example,electrokinetic or mechanical sorting into a pathway.

FIG. 1 shows an exemplary embodiment of an electrophoresis device. Asdepicted in FIG. 1, the arrangement can include a channel-definingmember 10 defining a channel 12 therein for the migration of an analytesample. The channel-defining member 10 can include a cover plate with orwithout grooves, an etched plate defining one or more capillary sizedgrooves therein, or one or more capillary tubes. According to variousembodiments, the channel-defining member can be an etched plate having aplurality of channels or grooves, or the channel-defining member caninclude a plurality of capillary tubes. The use of a plurality ofchannels can allow a large number of analyte samples to be measuredsimultaneously in order to increase throughput. As is well known, forelectrophoresis to occur, opposing ends of channel-defining member 10,such as an electrophoretic plate or capillary tube, can be placed incontact with corresponding electrodes connected to a power supply forgenerating an electric field across the plate or tube. This field cancause the analyte to migrate from a loading site (not shown) for theplate or tube arrangement of the channel-defining member 10, toward adetection site or detection zone 14. The detection zone can encompassthat zone on the channel that is irradiated by an irradiation source toexcite markers, such as dye markers, used to label analytes in thesample.

An example of a marker compound can be a dye marker. Any suitablemarker, such as, for example, a fluorophore, can be used. Fluorophoresuseful according to various embodiments can include those that can becoupled to organic molecules, particularly proteins and nucleic acids,and that can emit a detectable amount of radiation or light signal inresponse to excitation by an available excitation source. Suitablemarkers can encompass materials having fluorescent, phosphorescent,and/or other electromagnetic radiation emissions. Irradiation of themarkers can cause them to emit light at varying frequencies depending onthe type of marker used.

One class of markers provides signals for the detection of labeledextension and amplification products by fluorescence, chemiluminescence,or electrochemical luminescence (Kricka, L. in Nonisotopic DNA ProbeTechniques, Academic Press, San Diego, pp. 3-28 (1992)).Chemiluminescent labels can include 1,2-dioxetane compounds (U.S. Pat.No. 4,931,223; and Bronstein, Anal. Biochemistry 219:169-81 (1994)).Fluorescent dyes useful for labeling probes, primers, and nucleotide5′-triphosphates include fluoresceins, rhodamines (U.S. Pat. Nos.5,366,860; 5,936,087; and 6,051,719), cyanines (Kubista, WO 97/45539),and metal porphyrin complexes (Stanton, WO 88/04777). Fluorescentreporter dyes include xanthene compounds such as fluoresceins I andrhodamines II:

The ring positions of I and II above can be substituted. The amino Rgroups of II can be substituted. The substituents can include covalentattachments to the primers, probes, or nucleotides. Examples of formulaeI and II include wherein X can be phenyl substituted with carboxyl,chloro, and other groups, for example, as described in U.S. Pat. Nos.5,847,162; 6,025,505; 5,674,442; 5,188,934; 5,885,778; 6,008,379;6,020,481; and 5,936,087, which are incorporated herein in theirentireties by reference, and wherein X can be hydrogen, for example, asdescribed in U.S. Pat. No. 6,051,719, which is incorporated herein inits entirety by reference.

According to various embodiments, an optical instrument can be providedthat includes a light source arranged to emit an excitation wavelengthor wavelength range toward a region capable of retaining a sample, suchthat a fluorescent dye, if present in the region, can be caused tofluoresce. The light source can provide excitation wavelength rangesthat correspond to respective excitation wavelength ranges of aplurality of fluorescent dyes. A detector capable of detecting anemission wavelength emitted from a fluorescing dye can be used todetermine the absence or presence of a component associated with thedye. For example, the dyes can include intercalating dyes, reporterdyes, free-floating dyes, and the like.

According to various embodiments, PCR dyes can be used that onlyfluoresce when bound to a target molecule. Nucleic acid sequenceamplification dyes can also be attached to probes that also areconnected to quenchers, and the action of nucleic acid sequenceamplification enzymes will disassemble the dye-probe-quencher moleculecausing the dye to increase its fluorescence. According to variousembodiments, nucleic acid sequence amplification can be performed usinga variety of methods, for example, polymerase chain reaction (PCR),isothermal amplification reaction, well known in the art. When a PCRprocedure is used, for example, the number of unquenched dye moleculesdoubles with every thermal cycle. Fluorescing dyes are well known in theart, and any of a plurality of fluorescent dyes having variousexcitation wavelengths can be used. Examples of such dyes include, butare not limited to, Rhodamine, Fluoroscein, dye derivatives ofRhodamine, dye derivatives of Fluoroscein, 5-FAM™, 6-carboxyfluorescein(6-FAM™), VIC™, hexachloro-fluorescein (HEX™), tetrachloro-fluorescein(TET™), ROX™, and TAMRA™. Dyes or other identifiers that can be usedinclude, but are not limited to, fluorophores and phosphorescent dyes.Dyes can be used in combinations of two, three, four, or more dyes persample. According to various embodiments, the family of 5-FAM™, 6-FAM™,VIC™, TET™, and/or ROX™ dyes can be used to indicate the presence ofsample components.

According to various embodiments, various detectable markers can beused, in addition or in alternate, to dyes. Markers can include, forexample, fluorescing dyes, free-floating dyes, reporter dyes, probedyes, intercalating dyes, and molecular beacons. Dyes that fluorescewhen integrated into DNA can be intercalating dyes. Other dyes known as“reporter” dyes can attached to the ends of “probes” that have“quenchers” on the other end. A nucleic acid sequence amplificationreaction, for example, PCR, can result in the disassembly of theDye-Probe-Quencher molecule, so the reporter dye can emit an increasedamount of fluorescence. Reporter dyes are not attached in any way to thesample. Free floating dyes can be floating freely in solution. Otherfluorescing markers well know in the art can be utilized. According tovarious embodiments, molecular beacons can be single-stranded moleculeswith hairpins that preferentially hybridize with an amplified target tounfold. According to various embodiments, quantum dots can be used asmarkers also.

In the embodiment shown in FIG. 1, an irradiation source is providedthat emits excitation light in a given frequency range, such as, forexample, a light emitting diode (LED) 16. It can be to be noted that, inthe instant description, the source of light can be any of a variety oflight sources. According to various embodiments, the light can have afrequency of about 660 nm or lower. The irradiation source can be, forexample, an LED, an organic LED, a non-coherent light source as known tothose skilled in the art, a solid state laser, a microwire laser, or acombination thereof. As used herein, the terms “irradiation source,”“light source,” “excitation source,” “LED,” or the like can includesingle or multiple sources of irradiation, including LED flood lightarrays. As used herein, “LED” can refer to an LED, an OLED, ormultiplicities thereof. Further, according to various embodiments, the“LED” in can include coherent irradiation sources, for example, a solidstate laser or a micro-wire laser.

According to various embodiments, the light from the LED can bemodulated by an excitation modulating optics system before reaching thedetector zone 14. The excitation modulating optics system can include,as shown, a conditioning filter 18, the role of which can be tosubstantially block predetermined ranges of wavelengths of light emittedby the LED. The predetermined ranges can correspond to wavelengths oflight that can overlap with the emission spectra of the markers beingused. According to various embodiments, the conditioning filter can letthrough only light in the wavelength range of the excitation light ofone or more of the markers. Any given LED can emit excitation light in aspectral range. The range of wavelengths of the excitation light in turncan excite markers to emit light signals within a given spectral rangein the detection zone. For the detection of light signals from thedetection zone, that portion of the excitation light that would be inthe same wavelength range as some or all of the light signals emittedfrom the detection zone can be blocked. The light passing through theconditioning filter 18 is conditioned light 20, as seen in FIG. 1.

The excitation modulating optics system can further include a focusingoptical system 19. The conditioned light 20 can be focused by focusingoptical system 19 to irradiate the analyte sample and correspondingmarkers in the detection zone 14. The thus irradiated marker or markerscan turn emit light signals, such as through fluorescence, atfrequencies specific to the irradiated marker, so as to present a peakintensity. For example, a dye excited by yellow light can have afluorescence emission peak intensity at 610 nm corresponding to theorange portion of the spectrum. A peak intensity of about 460 nm can beassociated with the blue portion of the spectrum, and a peak intensityof about 660 nm can be associated with the red portion of the spectrum.By way of example, ROX, a known dye marker, can be best excited at 590nm. ROX can be excited by an LED emitting radiation at 590 nm.

The device of FIG. 1 can include a collection modulating optics systemthat can include a collimating optical system 24, a wide bandpass filter26, a transmission grating 28, and a re-imaging optical system 30.Emitted light 22 from the detection zone 14, and, in addition,conditioned light 20 passing through the detection zone 14, can becollimated by a first optical component or system 24. The light from thedetection zone can include the emitted light 22 and a portion of theconditioned light 20 passing through the detection zone. Alternatively,the excitation light can be brought in at an angle with respect to thedetection zone such that most of the conditioned light passing throughthe detection zone is not collected by the collimating optical system24. This reduces the excitation light that might be rejected. However,such an alternative arrangement also decreases the level of excitationlight that hits the detection zone 14. It can be, nevertheless, possibleto establish a compromise between irradiation angle and level ofexcitation light, as readily recognizable by those skilled in the art.

The light 20 and 22 from the detection zone 14 can be collimated bycollimating optical system 24. What is meant in the context of variousembodiments by collimation is any reduction in the propagation angle ofthe light being collimated. According to various embodiments, thereduction in the propagation angle of the light being collimated canresult in a propagation angle between about 20 degrees and about 0degrees.

According to various embodiments, a long pass filter, or, in thealternative, a wide bandpass filter 26, can be used for letting through,substantially exclusively, predetermined wavelengths of light from thedetection zone corresponding to a portion of wavelengths of the lightsignals emitted by an associated marker. The portion of the wavelengthsof the light signals can include, for example, all of the light signals,or it can include, for each marker, a range of wavelengths of the lightsignals, such as a range of wavelengths about the peak intensity of thelight signals. As an example, a wide bandpass filter can blockwavelengths of light outside of the range of from about 500 nm to about700 nm, thereby letting through only light that corresponds veryspecifically to the light emitted by the markers, that is, correspondingto emitted light 22. Thereafter, emitted light 22 can be spectrallydistributed by a transmission grating 28 and refocused by re-imagingoptical system 30 onto an array 34 of solid-state detectors, or detectorarray 34. The excitation modulating optics system and the collectionmodulating optics system are hereinafter collectively referred to as“the modulating optics system” or “modulating optics.” According tovarious embodiments, the array of solid-state detectors can include thephoto-detecting surface of the parallel register of a charge-coupleddevice (CCD) 32. As shown in FIG. 1, the image produced by refocusingemitted light 22 can be projected onto the detector array 34 of the CCD,producing a pattern of charge in proportion to the total integrated fluxincident on each pixel of the parallel register, as is well known in theart.

According to various embodiments, at least one of the excitation beampathway and the emission beam pathway can pass through a lens system.The lens system can include a ball lens, for example. The modulatingoptics system can include the lens system. For example, the excitationbeam pathway and/or the emission beam pathway can pass through a lenssystem as described in U.S. patent application Ser. No. 10/805,096,filed Mar. 19, 2004, which is incorporated herein in its entirety byreference. The modulating optics system can include or be separate froma lens system as described in U.S. patent application Ser. No.10/805,096.

Referring additionally now to FIGS. 2 and 3, an image produced by movinganalyte bands can be recorded by the photo-detecting surface of CCD 32(FIG. 1) at times t (FIG. 2) and t+Δt (FIG. 3). The photo-detectingsurface 36 can be part of the two dimensional detector array 34 shown inFIG. 1. Photo-detecting surface 36 can include a spectral axis asindicated by arrow λ on the figure, and a spatial axis along which theanalyte bands move, as indicated by arrow M, in FIGS. 2 and 3. Asfurther seen in FIG. 2, an image created by the light signals emitted byexcited markers can produce, for example, two bands 38 and 40 onphoto-detecting surface 36, for example, substantially in the red andblue regions of the spectrum, respectively. Each band can correspond toa marker used to label, for example, a predetermined type of analyte.The bands can be spectrally distributed along the spectral axis bytransmission grating 28 as shown in FIG. 1. At time t, as shown in FIG.2, the charges produced on surface 36 can present two respective peaks46 and 48 on intensity profile 45. These peaks can correspond to bands38 and 40, respectively. As seen in FIG. 3, at time t+Δt, both bands 38and 40 can have moved downward along the direction of migration M on thephoto-detecting surface 36. Serial register 42 of the CCD 32 (FIG. 1)collects the charges accumulated for each analyte band during itsintegration time. All signals received from the detector can beconverted from analog to digital, and conveyed to a serial port fortransmission to a multipurpose computer for storage and/or for furtherprocessing and analysis. The analog output can alternatively oradditionally be sent directly to an output device for display orprinting, or used for other purposes.

According to various embodiments, for example, as shown in theembodiment above, collection of the image can be performed using timedelay integration (TDI). In the CCD, the photogenerated charge in thephotoactive elements or pixels can be transferred toward the serialregister 42 one row at a time. The charge information in the serial rowcan be read by using a corresponding single on-chip amplifier or readoutregister 44 of the CCD. By way of example, for a 256×256 element CCD,each time a single imaging area (one row) is transferred to the serialregister 42, 256 readouts of the thus transferred area are performed,each readout corresponding to a different spectral element or pixel inthe row. The above process continues until all 256 pixels in all 256rows have been read.

Under a normal read-out approach, the motion of the images on thedetector array 34 produces a blur. In TDI, according to variousembodiments, the shutter can be eliminated. The shifting of rows of theCCD can be synchronized to the migration of the band of analyte in thechannel. Thus, as an analyte band enters the excitation zone of the LED(detection zone 14), the light signals emitted therefrom can becollected and can illuminate the first row of the CCD, and thecorresponding charge information can be read using the amplifier orreadout register 44 of the CCD. The band takes a period of time, Δtp, tomigrate in the channel so that its corresponding image migrates to thenext row of the CCD, one row closer to the serial register. After thistime period, the charge on the CCD can be shifted one row closer to theserial register, such that the fluorescence from the analyte correspondsto the same charge information on the CCD. Therefore, distinct from thephysical rows of the CCD, there exists in TDI according to variousembodiments a continuously moving row of accumulating photogeneratedcharge. An example of TDI in a capillary electrophoresis system usinglaser-induced fluorescence is disclosed in U.S. Pat. No. 5,141,609 toSweedler et al., and in J. F. Sweedler et al., Fluorescence Detection inCapillary Zone Electrophoresis Using a Charge-Coupled Device with TimeDelayed Integration, Anal. Chem. 63, 496-502 (1991), the contents ofboth of which are incorporated herein in their entireties by reference.

According to various embodiments, the effective integration time for agiven analyte band can vary from application to application. Theeffective integration time of a given analyte band can correspond to atime where the portion of the wavelengths of the light signals in theanalyte band being integrated moves across two pixels on the detectorarray, or to the entire time the portion of the wavelengths of the lightsignals in the analyte band being integrated is in the detection zone,or to any time therebetween. In addition, the portion of the wavelengthsof the light signals in the analyte band being integrated can, accordingto various embodiments, include (1) a range of wavelengths about a peakintensity of the light signals; (2) a range of wavelengths including allwavelengths of the light signals; or (3) a range of wavelengths anywherebetween (1) and (2) above. By way of example, the integration time caninclude a time it would take for the detector to integrate a range ofwavelengths of the analyte band corresponding to a full width of anintensity curve of the light signals in the analyte band at half of thepeak or maximum intensity of the intensity curve, or “full width at halfmax” of the intensity curve. The portion of the wavelengths of the lightsignals in the analyte band being integrated can depend on the number ofdifferent colors being integrated, and on how well the colors areseparated from one another in the emission spectra. As a general rule,the better separated the colors in the emission spectra, the wider theportion of the wavelengths of the light signals, and, hence, the longerthe effective integration time.

According to various embodiments, the use of TDI in collecting datapoints addresses the problem of lowered irradiance when usingirradiation sources emitting non-coherent light, such as LEDs. Theirradiance, that is, photons emitted per millimeters squared, can betypically about a thousand times lower in LEDs when compared with theirradiance of lasers. According to various embodiments, TDI addressesthe problem of lowered irradiance by allowing a longer period of timefor the integration of signals from excited markers. According tovarious embodiments, a broad detection zone can be used for TDI. In anon-TDI detection system, the detection zone can be typically about onetenth of a millimeter squared. When using TDI according to variousembodiments, the detection zone for one channel can be one hundred timeslarger, that is, about one millimeter squared, allowing a relativelylarger number of markers to be excited and a larger number of datapoints to be integrated into a detector. The various embodimentsdescribed herein can be equally applicable in instances where aplurality of channels are present, the detection zones of each of therespective channels being adapted to be irradiated by one or moreirradiation sources emitting light.

For the purpose of accumulating charges to effect TDI, instead ofshifting the charges on the CCD as a function of the migration of theanalyte bands, according to various embodiments, the CCD itself and/orthe image itself, that is, the light signals from the detection zone,can be moved as a function of migration of the analyte bands. The resultof such movement of the CCD and/or image can be the tracking of eachanalyte band by a continuously moving row of accumulating photogeneratedcharge on the CCD during the effective integration time of the analyteband. By way of example, to accomplish the desired result mentionedabove, appropriate motors, gearing, belt drives, control units and powersupplies can be used. For example, a linear actuator can be used totranslate the re-imaging optical system 30 and/or the CCD itself tominimize blurring. This can make the image stationary on the CCDthroughout the integration time.

According to various embodiments, the CCD can be a frame transfer CCD. Aframe transfer CCD has a parallel register that can include two CCDregisters arranged in tandem. The CCD register, or storage arrayadjacent to the serial register, can be covered with an opaque mask andcan provide temporary storage for charges during readout. The other CCDregister, or image array, identical in capacity to the storage array,can be used for imaging. After the image array is exposed to light, theresulting charges can be rapidly shifted in the parallel register up tothe storage array for subsequent readout. This shift operation typicallytakes a millisecond. While the masked storage array is being read, theimage array can integrate charge from the next image. See Charge-CoupledDevices for Quantitative Electronic Imaging, Photometrics Ltd. (1992),the content of which is incorporated herein in its entirety byreference.

To effect TDI, according to various embodiments, the modulating opticsas defined herein can be moved relative to a channel-defining member.For example, over a given detection zone, the modulating optics can bemoved along the detection zone at substantially the same speed as theanalyte for detection moves along the detection zone in thechannel-defining member. In this manner, the marker of a component ofthe analyte band can be repeatedly imaged on the same portion of thedetector, for example, a CCD, over the integration time. Alternately,the channel-defining member can be moved at the same speed but in anopposite direction from the movement of the analyte along thechannel-defining member. Movement of the channel-defining memberopposite the movement of the analyte to be detected can give theappearance of the analyte remaining stationary. Movement of thechannel-defining member opposite the direction of movement of theanalyte, in combination with movement of the modulating optics oppositethe direction of movement of the analyte to be detected, can be used incombination in order to accumulate data on all analytes in thechannel-defining member.

Referring now to FIGS. 4, 5 a, and 5 b, and according to variousembodiments, instead of one irradiation source, a plurality ofirradiation sources can be provided to excite marker compounds in asample. In the embodiment shown in FIGS. 4, 5 a, and 5 b, theirradiation sources can include four LEDs 50, 52, 54 and 56. The LEDscan be positioned so as to irradiate channel-defining member 58, whichcan define two channels in the form of, for example, two capillarytubes, as shown in FIG. 5 b. Each LED can emit non-coherent light in apredetermined range of wavelengths. For example, LED 50 and LED 52 canemit substantially blue light, LED 54 can emit substantially greenlight, and LED 56 can emit substantially yellow light. As the aboveexample shows, multiple LEDs can be used to increase the availablelight. For example, if LEDs 50 and 52 emit blue light, they can be usedsimultaneously, either continuously or in a pulsed fashion, in this wayincreasing the amount of available blue light to obtain a proportionalresponse in the associated markers. Although each type of marker usedcan ideally be excited by a different wavelength, LEDs of the optimumwavelength and power level for excitation of a given marker may not beavailable for each given application. Thus, different markers can beexcited by the same LED, according to various embodiments.

According to various embodiments, the modulating optics accordingdepicted in FIGS. 4, 5 a, and 5 b, can be comparable to the modulatingoptics in the embodiment shown in FIG. 1, with like components havingbeen labeled with like reference numerals. Thus, for each irradiationsource, a conditioning filter 18 and a focusing optical system 19 areprovided, it being understood that the respective conditioning filtersand focusing system for the respective irradiation sources are not,however, necessarily identical merely by virtue of the fact that theyhave been labeled with like reference numerals. As previously noted withrespect to FIG. 1, the function of each conditioning filter 18 can be tolet through only light in the wavelength range of excitation light forone or more of the markers. The conditioning filters 18 each cansubstantially block predetermined ranges of wavelengths of light emittedby the corresponding LED. The predetermined ranges correspond towavelengths of light that can overlap with the emission spectra of themarkers being excited by the corresponding LED. Each focusing opticalsystem 19 can focus the conditioned light from the conditioning filteronto the detection zone 14. As shown, for example, in FIGS. 4, 5 a, and5 b, the detection zone 14 can correspond to a respective detection zonefor each of the shown capillary tubes. Excited markers in detection zone14 can emit light signals in the form of emitted light 22. The lightfrom the detection zone can include the emitted light 22, and, inaddition, a portion of the conditioned light 20 passing through thedetection zone.

According to various embodiments, the light 20/22 from the detectionzone can be collimated by collimating optical system 24, as shown, forexample, in FIG. 5 a. The collimated light can be passed through acorresponding bandpass filter 51 on filter wheel 60 as shown in brokenlines in FIGS. 5 a and 5 b. It can be noted that the bandpass filters51, 53, 55, 57, and FD, in FIGS. 5 a and 5 b have been shown in brokenlines because, in those figures, the depiction of the filter wheel 60 isnot cross-sectional, but rather represents plan views thereof.

Referring to FIG. 4, the filter wheel 60 is shown in more detail, andincludes a plurality of bandpass filters 51, 53, 55, 57, and FD. Each ofthe bandpass filters can be adapted to let through, substantiallyexclusively, predetermined wavelengths of light from the detection zonecorresponding to a portion of the wavelengths of the light signalsemitted by an associated marker. According to various embodiments, therecan be a bandpass filter provided for each associated marker. Theportion of the wavelengths of the light signals allowed through arespective bandpass filter can include all of the light signals emittedby an associated marker, or it can include a range of wavelengths aboutthe peak intensity of the light signals emitted by the associatedmarker. For example, the range of wavelengths about the peak intensityof emitted light signals can be between about 5% and about 20% ofwavelengths on each side of the peak intensity for a given marker, or itcan include the range of wavelengths at about half of the peakintensity, full width at half max. For example, as shown in FIGS. 4, 5a, and 5 b, bandpass filter 51 can be adapted to filter therethroughlight signals emitted by given markers responsive to LED 50. In FIGS. 5a and 5 b, the apparatus can be depicted in a mode where LED 50 canirradiate the detection zone 14. However, any of the shown LEDs 50, 52,54, and 56, can be selectively used to irradiate the detection zone 14,or the detection zone 14 can be irradiated by more than one LED. Thefilter wheel 60 can be actuated by a filter wheel mechanism 62, as shownin FIG. 5 a. The filter wheel mechanism 62 can control the filter wheelto selectively position, in the path of the collimated light, thebandpass filter corresponding to the marker excited by the active LED,that is, by the LED being used to irradiate the detection zone. Filterwheel mechanism 62 can be controlled by a microprocessor or othersimilar device (not shown) as known to those skilled in the art. Thefiltered light can be focused by a re-imaging optical system 30 onto anarray 34 of solid-state detectors, or detector array 34, for example,the photo-detecting surface of the parallel register of a charge-coupleddevice, for example, CCD 32. According to various embodiments, a singleLED can be used to excite all markers, or multiple LEDs can be used withone LED exciting each respective marker, or multiple LEDs can be used toexcite a marker, or combinations thereof. The selection and number ofbandpass filters for use can be a function of the markers themselves,the number of LEDs, or other factors as knows to those skilled in theart.

According to various embodiments, and as shown in FIG. 4, the filterwheel can include a filter FD thereon, adapted to let through only lightsignals generated by a fifth marker. Four markers can be used to labelthe moving analytes where the analytes are DNA fragments, each markercorresponding to a given one of the bases in a DNA chain, that is,purines A (adenine) and G (guanine), and pyrimidines C (cytosine) and T(thymine). According to various embodiments, a fifth marker can, forexample, be used for fragment analysis of the analytes. The fifth markercan be any marker, such as a dye marker, for doing fragment analysis asknown to those skilled in the art. It is to be noted that, according tovarious embodiments, the number of markers that can be used are notlimited to four or five as stated in the above example, but are ratherlimited only by the number of dyes available on the market andresponsive to the irradiation source or sources being used based onapplication needs. Fragment analysis can be accomplished using, forexample, the GENESCAN® Analysis software produced by Applied Biosystems,Inc., Foster City, Calif. The GENESCAN® Analysis software calculates thesize of the unknown analytes by generating a calibration or sizing curvebased upon the migration times of the analytes in a standard that havebeen labeled with a marker. The unknown analytes can be mapped onto thecurve and converted from migration times to sizes. In the case of theembodiment shown in FIG. 4, the fifth marker filter FD on filter wheel60 can let through light signals corresponding to markers used to labelanalytes in the standard. These markers can be excitable by at least oneof the irradiation sources 50, 52, 54, and 56, appropriately mounted toallow fragment analysis.

FIGS. 6 a through 6 e depict images on the detector array 34 of CCD 32shown in the embodiment of FIGS. 4, 5 a, and 5 b, wherein the images canbe produced by moving analyte bands. Exemplary images are shown forexemplary markers that can be used to label the analytes, and that canbe responsive to excitation by a given one of the irradiation sources50, 52, 54, and 56. Each frame of photo-detecting surface 36 shown inFIGS. 6 a through 6 e depicts two lanes of analyte bands, each lanecorresponding to one of the two capillaries of channel-defining member58. The bands move along the direction of migration M. The bands areshown in each of FIGS. 6 a through 6 e as being limited in the spectraldirection because the light recovered from the markers has been filteredthrough a corresponding bandpass filter 51, 53, 55, 57, or FD. At theright of each frame is an intensity profile 45 corresponding to chargeson the right lane of photo-detecting surface 36, which corresponds to acapillary of channel-defining member 58.

According to various embodiments, the intensity profiles can be alignedand combined according to known methods. The intensity profiles can bemulticomponented in order to account for any spectral overlap. Asdescribed with regard to the embodiment of FIG. 1, the serial registerof the CCD 32 in the embodiments depicted in FIGS. 4, 5 a, and 5 b,collects the charges accumulated for each analyte band during itsintegration time. All signals received from the detector can beconverted from analog to digital and conveyed to a serial port fortransmission to a multipurpose computer for storage, further processing,and analysis. Alternatively or additionally, the analog output can besent directly to an output device for display or printing. By way ofexample, a multipurpose computer can be used to perform themulticomponenting process. Multicomponenting is a process that is knownto those skilled in the art, and can involve a spectral calibrationwithin a multicomponenting software program. The spectral calibrationcan be obtained through a predetermined signature matrix correspondingto each marker. Each signature matrix can provide a signature snapshotof the intensity of light signals from a given marker as a function ofthe wavelengths of those light signals. By virtue of the signaturematrices, a combination of intensity curves for a given wavelength bandemitted from the detection zone can be broken down into its componentscorresponding to light signals emitted by individual ones of themarkers. In this way, a relatively accurate assessment of the lightsignals corresponding to respective ones of the markers can be made forthe detection process.

According to various embodiments, the apparatus as shown, for example,in FIGS. 4, 5 a, and 5 b, can irradiate detection zone 14 by irradiatingeach respective one of the irradiation sources 50, 52, 54, and 56, insequence. The filter wheel can be adjusted to dispose a bandpass filter51, 53, 55, 57, or FD, corresponding to the marker being used beforecollimating optical system 24. The detection zone can be irradiated forthe duration of the integration time, during which the analyte bandsmove across the detection zone. During the integration time, the chargesgenerated by the light signals from the markers to be detected can bemoved along a parallel register in the direction of migration. Thecharges can be accumulated in the detector or CCD 32 before they areread. Thereafter, the process can be repeated until all of theirradiation sources have irradiated the detection zone, and until allfilters, including filter FD, have been positioned before thecollimating lens to filter the light therefrom. Detector 32 can be aframe transfer CCD, wherein each frame of the CCD upon which chargeshave been accumulated can be transferred to a storage array, making theimage array available for the next series of charge accumulationsproduced by the next respective one of the irradiation sources beingused.

The above process can be repeated in cycles as many times as necessaryin order to obtain sufficient data regarding each analyte beingdetected. Fewer cycles can result in an increase in signal, becausefewer cycles mean longer integration times, and therefore increasedreadout signals over the noise typically associated with a CCD. On theother hand, increasing the number of cycles can improve the dynamicrange of the system. The dynamic range of the system is defined as thelargest peak signal that can be read by a given CCD (or “full wellcapacity”) over the smallest peak that can be read by the CCD just abovethe noise level. A CCD typically has a given full well capacity. If apeak signal is above the full well capacity of a CCD, it will be off thescale of the CCD. Short integration times allow peak signals to begenerally attenuated so as to reduce the possibility of saturating theCCD with off-scale signals, that is, with signals that go beyond theCCDs full well capacity. By using more cycles, analyte concentrationscan be increased while still allowing the CCD to reliably detect signallevels without saturation. According to various embodiments, there is atrade-off between using fewer cycles at a longer integration time suchas, for example, 5 seconds, and using more cycles at a shorterintegration time such as, for example, 1 second. Longer integrationtimes are useful where the noise level is relatively high and thesensitivity of the system needs to be increased because of the noiselevel. In systems where the noise level is relatively low, shorterintegration times allow multiple reads of signals from the same marker,and the read signals can be multicomponented, allowing the detection ofbrighter peaks without going off the scale of the CCD.

By way of example, a frame transfer CCD can collect light signalscorresponding to a blue marker during integration time t while an LEDexciting primarily the blue marker irradiates the detection zone.Thereafter, the entire CCD is read out. A filter wheel can be moved toposition a bandpass filter associated with a green marker before acollimating optical system, and an LED exciting primarily the greenmarker can irradiate the detection zone. The CCD can collect the lightsignals corresponding to the green marker during integration time t. Theentire CCD is then read out. The filter wheel can be moved to position abandpass filter associated with a yellow marker before a collimatingoptical system, and an LED exciting primarily the yellow marker canirradiate the detection zone. The CCD then collects the light signalscorresponding to the yellow marker during integration time t, and theentire CCD is thereafter read out. The above process can be repeated forall five markers, for example, as shown in FIGS. 4, 5 a, and 5 b. Theprocess can be repeated a number of times equal to the number ofmarkers. According to various embodiments, one or both of the LED andbandpass filter can be changed between CCD readouts. As suggested in theabove example, the image of the analyte band can take about five timesthe integration time to travel from the top of the frame transfer CCD tothe bottom thereof, that is, to the readout register. Each readout ofthe CCD can correspond to one marker. All of the readouts can be alignedand combined in a known manner for multicomponenting.

An example of multicomponenting is shown in FIGS. 6 f through 6 h. Forpurposes of FIGS. 6 f through 6 h, the filters 51, 53, 55, 57, and FD,are presumed to pass wavelengths of light in the blue, green, yellow,red and “fifth” portions of the spectrum, respectively. The “fifth”portion can, for example, be in the orange range of the spectrum. FIG. 6f is a schematic representation of an electropherogram showingfluorescence intensity curves during three readings of the signals bydetector 32, wherein each curve corresponds to light passed through arespective filter of the filter wheel in FIG. 4. The intensity curvescorrespond to a reading of light signals emitted from an analyte labeledwith a marker, for example, FAM, a dye marker that emits light signalsmostly in blue. The first portion of each curve, drawn in solid lines,corresponds to a reading from the respective blue filter, green filter,yellow filter, red filter, or fifth filter of FIG. 4 during a firstintegration time t. The second portion of each curve, drawn in brokenlines, corresponds to a reading from the respective filter during asecond integration time t. The third portion of each curve, drawn insolid lines, corresponds to a reading from the respective filter duringa third integration time t. In FIG. 6 f, the horizontal axis correspondsto distance traveled by the analyte, and the vertical axis corresponds,for each filter, to the fluorescence intensity of light emerging fromthat filter. Thus, the first set of curves in solid lines representsintensity curves for light passing through each filter during a firstcycle of the filter wheel 60. The second set of curves in broken linesrepresents intensity curves for light passing through each filter duringa second cycle of the filter wheel 60. The third set of curves in solidlines represents intensity curves for light passing through each filterduring a third cycle of the filter wheel 60. As seen in FIG. 6 f, thelight from the blue filter exhibits the most intensity during eachcycle, the intensity decreasing as light is collected from the greenfilter, the yellow filter, the red filter and the fifth filter,respectively. As seen in the instant example, therefore, spectraloverlap causes FAM to emit mostly in blue, some in green, less inyellow, etc. As filter wheel 60 is rotated within each cycle, to place asubsequent filter in the path of fluorescence from the detection zone14, the analyte moves a distance x as marked on FIG. 6 f.

FIG. 6 g is a schematic representation of the intensity curves of FIG. 6f in an aligned format for multicomponenting. Each intensity curve otherthan the one corresponding to blue light can be shifted by a multiple ofx, x being a function of the filter to be aligned with the intensitycurve corresponding to blue light. After being aligned, the intensitycurves are combined and multicomponented, yielding the intensity curvefor FAM shown in FIG. 6 h. To the extent that only FAM is being detectedin the example of FIGS. 6 f through 6 h, the intensity curves for markerdyes JOE, TAMRA, ROX, and the fifth dye, are shown as flat in FIG. 6 h.

FIGS. 6 i and 6 j demonstrate representative excitation efficiencycurves and fluorescence intensity curves, respectively, plotted versuswavelength for four different dye markers that can be used inoligosynthesis, namely, 5-FAM, JOE, TAMRA, and ROX. These dye markersare exemplary of those that can be used in various embodiments where aplurality of dye markers are to be used, such as with systems shown inFIGS. 4, 5 a, 5 b, and 7.

In FIG. 6 i, the x-axis corresponds to wavelengths, expressed innanometers (nm), emitted by an irradiation source. The y-axiscorresponds to the percentage of excitation efficiency. As shown in FIG.6 i, 5-FAM has a maximum absorbance, corresponding to its peak percentexcitation efficiency, at about 490 nm. When maximum absorbance of a dyemarker occurs at a given wavelength, it indicates that the dye markerfluoresces at its peak fluorescence intensity when it is irradiated atthat given wavelength. As further shown in FIG. 6 i, JOE has a maximumabsorbance at about 526 nm, TAMRA has a maximum absorbance at about 560nm, and ROX has a maximum absorbance at about 588 nm. FIG. 6 i alsoshows that, when a dye marker, such as 5-FAM, is irradiated at itsmaximum absorbance wavelength, other dye markers, such as, for example,JOE, TAMRA, and ROX, do exhibit some absorbance, although to a lesserextent than 5-FAM. Any irradiation source, for example, an LED, can beused to emit the wavelengths indicated on the x-axis.

In FIG. 6 j, the x-axis corresponds to wavelengths of fluorescent light,expressed in nm, emitted by excited dye markers. The y-axis correspondsto the percentage of fluorescence intensity. As seen in FIG. 6 j, 5-FAMhas a peak fluorescence intensity at about 522 nm, JOE has a peakfluorescence intensity at about 554 nm, TAMRA has a peak fluorescenceintensity at about 582 nm, and ROX has a peak fluorescence at about 608nm. The wavelengths on the x-axis are those that can be emitted by thefour mentioned dye markers. FIG. 6 j shows that where a dye marker, suchas TAMRA, fluoresces at its peak fluorescence intensity, other dyemakers, such as 5-FAM, JOE, and ROX, also fluoresce, although at lesserfluorescence intensities than TAMRA.

When different light wavelength sources are used, each dye marker in thedetection zone can be excited efficiently, and in a way that allowsdetection by its unique spectral signature. When two dye markers exhibitfluorescence intensity peaks that are close together, for example, whenthe difference between the fluorescence intensity peaks of the two dyemarkers is less than about 30 nm, there can be a high level of overlapof the light emitted by the two dye markers. A high level of overlap canmake it difficult to distinguish between the light emitted by the twodye markers, and therefore can make it difficult to determine therelative amounts of the two dye markers. Typically, there is overlappresent in the excitation and emission wavelengths of the different dyemarkers, as suggested, for example, in FIGS. 6 i and 6 j. However, theoverlap can be minimized by selecting dye markers that present easilydistinguishable fluorescence intensity peaks, such as those shown inFIG. 6 j. According to various embodiments, it is possible to firststart with an irradiation source emitting radiation within a given rangeof wavelengths, and to investigate each such irradiation source to seehow well it excites available dye markers. Graphs such as those shown inFIGS. 6 i and 6 j can be used in this context. For example, whereexcitation efficiency curves of various dye markers are plotted as shownin FIG. 6 i, an irradiation source, such as an LED emitting light in agiven range of wavelengths, can be predicted to excite given ones of thedye markers based on their excitation efficiencies. Using a fluorescenceintensity graph as shown in FIG. 6 j, the amount of overlap betweenintensity peaks of the different markers can be determined. Using thisinformation, a set of dye markers best suited for a particularapplication can be chosen. Once the dye makers are chosen, the filterscorresponding thereto can be chosen, for example, the filters shown onthe filter wheel of FIG. 4 described above, and on the filter wheel ofFIG. 7 described below. The best set of markers can exhibit the desiredminimum amount of overlap at the emitted wavelengths that are to bedetected.

Because each marker can have a different excitation curve, thefluorescence output of a marker can be dramatically increased by the useof an LED that is well matched to the excitation wavelength of themarker. Matching the light source and marker can increase emission fromthe marker of interest, while minimizing undesired light, for example,background light from the system and emissions from other markers,resulting in better quality data. For example, if a blue/green LEDhaving an emission wavelength of about 503 nm is used for the markerdesignated FAM, the excitation of FAM can be high, about 80%, and theexcitation for another marker, for example, ROX, can be low, forexample, about 6%. Similarly, a yellow LED with an emission wavelengthof about 592 nm will not excite FAM, but can excite ROX in an amount ofabout 90%.

Another embodiment of an apparatus is depicted in FIG. 7. As in theembodiments shown in FIGS. 4, 5 a, and 5 b, a number of irradiationsources can be used to sequentially irradiate a detection zone. Thegenerated light signals having wavelengths in differing frequency rangescan correspond to charges spatially offset on the same array of thedetector, wherein the offset is a function of the bandpass filter usedin connection with the markers emitting the light signals. Reading ofthe accumulated charges on the detector array during time delayintegration (TDI) can be continuous rather than a frame-by-framereading.

As shown in FIG. 7, the irradiation sources, together with associatedoptics such as the conditioning filter 18 and the optical system forfocusing 19, can be provided on an irradiation source wheel 61.Components of the apparatus of FIG. 7 that are similar to those in FIG.1 have been labeled with the same reference numerals, such asconditioning filter 18, focusing optical system 19, collimating opticalcomponent or system 24, and re-imaging optical component or system 30.An irradiation wheel 61 can be rotatable to selectively position eachrespective one of the irradiation sources and associated optics toirradiate a detection zone 14. Any number of irradiation sources, forexample, four irradiation sources 50, 52, 54, and 56, as shown, forexample, in FIGS. 4, 5 a, and 5 b, can be provided. A filter wheel 61′can be provided, similar to filter wheel 60 in FIGS. 4, 5 a, and 5 b.The rotation of both wheels 61 and 61′ can be effected by the provisionof a filter wheel drive 62 similar to the filter wheel drive of FIGS. 4,5 a, and 5 b, described above. According to various embodiments, the twowheels 61 and 61′ can be coupled to one another and to the filter wheeldrive 62 by way of a rotatable shaft 63. According to variousembodiments, a plurality of irradiation sources can be provided that arenot on an irradiation wheel. According to various embodiments, the twowheels 61 and 61′ can be actuated independently by their own respectivewheel drives, and are not coupled to one another by a shaft.

As shown in FIG. 7, the apparatus can be provided with an offset system64. Offset system 64 can either be disposed on the filter wheel 61′ inassociation with a corresponding bandpass filter, or can be coupled toat least one of a detector 32, one or more components of the modulatingoptics system, and the detection zone 14. The offset system 64 canspatially offset the light signals impinging upon the array 34 of thedetector 32 by a predetermined amount as a function of the bandpassfilter being used. The offset can be accomplished by an offset system 64including a plurality of offset mechanisms 66 disposed on filter wheel61′, wherein the offset mechanism 66 is shown in broken lines in FIG. 7.Each offset mechanism 66 can be associated with a respective one of thebandpass filters to offset the filtered light therefrom. The offsetmechanisms 66 can include one or more grating, mirror, prism, or anyother device for offsetting light as known to those skilled in the art,or a combination thereof. One or more offset mechanism 66 can bedistributed about the circumference of filter wheel 61′ adjacent thecorresponding bandpass filter, and between the bandpass filter and thedetector 32, otherwise being identical to wheel 60 in FIG. 4. In thealternative, the offset can be accomplished by providing an offsetsystem 64 including an offset control device 67, also shown in brokenlines in FIG. 7. The offset control device 67 can be coupled to at leastone of the detection zone 14, one or more component of the modulatingoptics system, and the detector array 34, in order to offset the lightsignals impinging upon the array 34 of the detector 32.

According to various embodiments, the offset system can move at leastone of the detection zone 14, collimating optical system 24, re-imagingoptical system 30, or detector array 34, by a predetermined amount inorder to offset the light signals impinging upon the detector array 34.The offset system can include any suitable device for effecting atranslational movement of at least one of the detection zone 14,collimating optical system 24, re-imaging optical system 30, or detectorarray 34, as known to those skilled in the art. Such devices caninclude, for example, solenoids, or motor driven linear actuators, forexample, lead screws, rack-and pinion systems, cams, and the like. Forexample, a cam can be attached to drive shaft 63 to cause apredetermined translation of the re-imaging optical system 30 in anumber of ways recognizable by those skilled in the art. Thepredetermined amount of translation can correspond in a one to one ratiowith the amount by which the light signals impinging upon the detectorarray are sought to be offset. The range of wavelengths offset by thepredetermined amount can be a function of the irradiation source, themarkers, or other factors known to those skilled in the art.

The predetermined amount by which a given set of light signalscorresponding to an irradiation source and its respective bandpassfilter is to be offset can be readily determined by determining where onthe detector array the charges produced by the given set of lightsignals corresponding to a specific bandpass filter should be situatedwith respect to the detector array itself, and with respect to sets oflight signals in different wavelength frequency ranges corresponding tothe other bandpass filters. Thus, where individual mechanisms 66 areused in conjunction with a corresponding bandpass filter to offset thelight emerging therefrom, each mechanism 66 can be chosen according tothe frequency range of wavelengths that the bandpass filter letsthrough. In the alternative, where offset control device 67 is used, theoffset control can be programmed to offset at least one of the detectionzone 14, one or more components of the modulating optics system, and thedetector array 34, with respect to one another by the predeterminedamount. The offsetting could, by way of example, be accomplished bymoving the detection zone 14, the modulating optics, or the detectorarray 34, in a translational motion by the predetermined amount, causingthe image created by the light signals to be correspondingly spatiallyoffset. The embodiment shown in FIG. 7 can involve the use of aplurality of LEDs similar to those used in the embodiment of FIGS. 4, 5a, and 5 b. The offset amount can be sufficient be prevent overlap ofthe images from each bandpass filter.

Offset system 64, including offset mechanisms 66, or, in thealternative, offset control device 67, is shown in broken lines in FIG.7 in order to suggest that mechanisms 66 or offset control device 67 canbe used as alternatives for the offset system 64. According to variousembodiments, both alternatives, that is, mechanisms 66 and offsetcontrol device 67, can be used in conjunction with one another. Themodulating optics can include at least one of conditioning filter 18,focusing optical system 19, collimating optical system 24, andre-imaging optical system 30, or any devices or systems known to achievethe functions associated with the components listed above as known tothose skilled in the art.

According to various embodiments, the detection zone can include anysuitable channel-defining member, for example, any number ofcapillaries, or any number of channels, in, for example, an etchedplate. The channel-defining member can be a slab plate. According tovarious embodiments, the channel-defining member can assume anyorientation according to application needs, for example, a horizontalorientation or a vertical orientation.

According to various embodiments, any suitable number of bandpassfilters can be used, the number being determined at least in part by themarkers being used. Any number of different irradiation sources, forexample, LEDs, that can emit light in any number of wavelength ranges,can be used. The number of LEDs can be dependent, in part, on the amountof available space, the availability of LEDs corresponding to theexcitation wavelength ranges of the marker(s), and/or the type ofmarker(s) that can be used.

As used herein, the term “optical system” can include a single lens, alens system, a mirror system, or any other optical system capable offulfilling the desired and stated functions, as readily recognizable bythose skilled in the art.

In operation, the detection zone 14 in FIG. 7 is irradiated by a firstone of the irradiation sources, for example, by LED 50, as depicted inFIG. 7. The light signals emitted by the markers excitable by the lightfrom LED 50 can be, as previously described, filtered through acorresponding bandpass filter 51, and thereafter focused onto detectorarray 34 by a re-imaging optical system. Where offset system 64 includesmechanism 66, the filtered light from bandpass filter 51 is offset by arespective mechanism 66 by the predetermined amount corresponding to therange of wavelengths that the bandpass filter lets through, as describedabove. Each subsequent irradiation source and corresponding bandpassfilter can be positioned to irradiate the detection zone and to filterthe light therefrom through a rotation of the wheels 61 and 61′ inconjunction with one another. As previously suggested, any suitablenumber of markers, bandpass filters, and LEDs can be used in the system.

In the embodiment of FIG. 7, charges can be accumulated for eachrespective irradiation source during the integration time of the analytebands excitable by light from the respective irradiation source. Theaccumulation of charges can be effected, as previously described, byshifting the charges on the detector array, by moving relative to oneanother the detector array and light signals from the detection zone, bymoving the modulating optics relative to the channel-defining member, ora combination thereof. After each integration time, the wheels 61 and61′ can be rotated to position the next irradiation source andcorresponding bandpass filter in a functional position, that is, in aposition for the irradiation source to irradiate the detection zone andfor the bandpass filter to filter the light from the detection zone. Theprocess can be repeated until one cycle is completed, that is, until allof the irradiation sources and filters have been used once. The cyclecan be repeated as many times as necessary and/or desired on anapplication-by-application basis. To the extent that the exemplaryembodiment of FIG. 7 includes an offset system for spatially offsettinglight signals in differing frequency ranges, the exemplary embodimentcan allow a continuous reading of accumulated charges by the detector,thereby making possible a continuous time delay integration of the lightsignals from the analytes into the detector array 34. Alternately, asshown in the exemplary embodiments of FIGS. 4, 5 a, and 5 b, theaccumulated charges corresponding to each range of wavelengths of lightsignals can be read and discarded by the detector before charges for thenext range of wavelengths are read.

FIG. 8 depicts an exemplary image that can be produced on the array ofdetector (CCD) 32 by the moving analyte bands in each of the twocapillaries 58 for each of the wavelength ranges. As depicted in theimage, each range of wavelengths is assigned an arbitrary color, forexample, “blue,” “green,” “yellow,” “red,” and “fifth.” For each color,the column on the left corresponds to charges produced by light signalsfrom one capillary, and the column on the right corresponds to chargesproduced by the second capillary. The array depicts charges generated bylight signals across the color axis λ, offset with respect to oneanother by offset system 64 in the manner previously described.According to various embodiments, the number of wavelength ranges usedcan be dependent on the particular application, and can range from oneto as many as the system supports. The λ axis is referred to here as the“color axis” rather than the “spectral axis,” because the colors, onefor each bandpass filter, do not need to be arranged from shorter tolonger wavelengths. According to various embodiments and as shown inFIG. 8, to the extent that charges from light signals of differingwavelengths can be produced on the same array of light signals so as tobe spatially offset with respect to one another, those charges can beaccumulated during the integration time and read by the detector on acontinuous basis. This can eliminate the need to shut off the detectorin order to allow a reading of charges in a given range of wavelengthson a frame-by-frame basis, and/or can eliminate the need for a frametransfer CCD. Further, a longer integration time and simpler data outputcan be achieved, and a cheaper CCD can be used.

According to various embodiments, the detection zone, including one ormore channels, or a slab gel, can be irradiated with multiple colorirradiation sources, for example, multiple color LEDs. The use ofmultiple color LEDs can greatly improve the absorption efficiency ofsome of the markers. According to various embodiments, an irradiationsource can be conditioned to provide only a narrow wavelength range oflight. A bandpass filter can be used to substantially block all of theunwanted excitation light from the irradiation source. As used herein,“irradiation source” can include one or more sources of irradiation. Anexample of the results, in the form of graphs demonstrating the effectsof a conditioned irradiation source in combination with a bandpassfilter, are illustrated in FIGS. 9 a through 9 d.

According to various embodiments and as depicted in FIGS. 9 a through 9d, a detection zone of an apparatus can be irradiated by two irradiationsources simultaneously, although any number of irradiation sources canbe used simultaneously according to various embodiments. A system suchas the one shown in FIG. 1 can be used, replacing the single LED in FIG.1 with a set of two or more LEDs. The associated optics can be alteredaccording to the number of irradiation sources used.

According to various embodiments, a conditioning filter can be used foreach set of LEDs. The conditioning filter can substantially blockpredetermined ranges of wavelengths of light emitted by the set of LEDsas previously described, each predetermined range corresponding to arespective LED, as demonstrated, for example, in FIG. 9 a. FIG. 9 ashows a graph of relative excitation intensity for each of two LEDs of aset of LEDs, versus wavelength expressed in nanometers. The graph ofFIG. 9 a depicts the two LEDs as being in the violet and orange portionsof the spectrum, respectively. It is to be understood that variousembodiments are not limited to the above two types of LEDs, butencompasses any number of LEDs emitting light in any range offrequencies according to application needs. As depicted in FIG. 9 b,each predetermined range of wavelengths that passes through theconditioning filter corresponds to a subset of the wavelengths emittedby each respective LED depicted in the graph of FIG. 9 a. Thewavelengths that pass through the conditioning filter can be capable ofexciting the markers responsive to each respective LED. FIG. 9 b is agraph of percent transmission of light through the conditioning filterversus wavelength. As shown in FIG. 9 b, the conditioning filter cansubstantially block all excitation light except for wavelengths aroundthe respective LED emission maximum. For example, the conditioned lightranges can correspond to a wavelength range of from about 450 nm toabout 490 nm, and to a second wavelength range of from about 580 nm toabout 605 nm, corresponding to the first and second irradiation sources,respectively. The conditioning filter, and the predetermined rangescapable of passing therethrough, can be functions of the LED set beingused.

According to various embodiments, the conditioning filter can include asingle conditioning filter, or a series of conditioning filters, capableof filtering the light from the set of LEDs. The conditioned light canbe focused onto the detection zone of an electrophoretic detectionsystem, as previously described above. The light emitted by markers inthe detection zone can be passed through a bandpass filter. The bandpassfilter can allow, substantially exclusively, predetermined wavelengthsof light from the detection zone to pass through, wherein thepredetermined wavelengths of light correspond to a portion of thewavelengths of the light signals emitted by an associated set ofmarkers. The light that passes through the bandpass filter can befiltered light. The bandpass filter can substantially block theexcitation light from the LEDs which passes the detection zone. Theportion of the wavelengths of the light signals that can pass throughthe bandpass filter can include all of the light signals, or a range ofwavelengths about the peak intensity of light signals of each respectivemarker. For example, the range of wavelengths about the peak intensityof light signals can be between about 5% and about 20% of wavelengths oneach side of the peak wavelength of a given marker, or it can includefull width at half max. The collection of the predetermined wavelengthscan be by dispersion, for example, as shown in FIG. 1, or by use ofadditional bandpass filters, as shown, for example, in FIG. 4.

According to various embodiments, a conditioning filter can be usedbetween the irradiation source (light source) and the detection zoneholding a sample solution. The beam emitted by the light source is alsoknown as an excitation beam that can be filtered by an excitation filterat an excitation wavelength range. Comparably, a conditioning filter canbe used between the detection zone holding the sample solution and adetector. The fluorescence or luminance emitted by the sample uponexcitation is also known as an emission beam that can be filtered by anemission filter at an emission wavelength range.

According to various embodiments, a light source can be used to provideexcitation beams to irradiate a sample solution containing one or moredyes. For example, two or more excitation beams having the same ordifferent wavelength emissions can be used such that each excitationbeam excites a different respective dye in the sample. The excitationbeam can be aimed from the light source directly at the sample, througha wall of a sample container containing the sample, or can be conveyedby various optical systems to the sample. An optical system can includeone or more of, for example, a mirror, a beam splitter, a fiber optic, alight guide, or combinations thereof.

According to various embodiments, one or more filters, for example, abandpass filter, can be used with a light source to control thewavelength of an excitation beam. One or more filters can be used tocontrol the wavelength of an emission beam emitted from an excited orother luminescent marker. One or more excitation filters can beassociated with a light source to form the excitation beam. One or morefilters can be located between the one or more light sources and asample. One or more emission filters can be associated with an emissionbeam from an excited dye. One or more filters can be located between thesample and one or more emission beam detectors.

According to various embodiments, one or more filters, for example, abandpass filter, can be used with a light source to control thewavelength of an excitation beam. One or more filters can be used tocontrol the wavelength of an emission beam emitted from an excited orother luminescent marker. One or more excitation filters can beassociated with one or more light sources to form at least oneexcitation beam. One or more filters can be located between the one ormore light sources and a sample. According to various embodiments, oneor more emission filters can be associated with an emission beam from anexcited dye. One or more filters can be located between the sample andone or more emission beam detectors.

According to various embodiments, a filter can be used that is a singlebandpass filter or a multiple bandpass filter. As used herein, abandpass filter and a passband filter are used interchangeably. Amultiple passband filter can be, for example, a multiple-notch filter ora multi-Rugate filter. A multiple passband filter can be used with anincoherent light source, for example, a halogen lamp, a white lightsource, and/or one or more LEDs or OLEDs emitting light at differentwavelengths. A multiple passband filter can be used with a multiplelaser-based light source emitting light at different wavelengths.Examples of manufacturing and use of Rugate filters and Rugate beamsplitters can be found in, for example, U.S. Pat. No. 6,256,148 toGasworth which is incorporated herein by reference in its entirety.

According to various embodiments, a multiple passband filter can be usedwith a dichroic beam splitter, a 50/50 beam splitter, a dichroic beamsplitter that has several “passbands,” or no beam splitter. A multiplebeam splitter can be coated at an angle, causing a variance in athickness across a filter substrate, to compensate for wavelength shiftwith an angle. A beam splitter can be disposed at a low angle along alight or beam path to sharpen edges of the filtered light and can easefabrication. A beam splitter can be disposed at a 45° angle of incidencealong a light beam path. A low angle can include an angle of incidenceless than 45°, less than 30°, or less than 15°. A multiple passbandfilter can be formed by coating different light interference materialsover respective areas of a substrate used in a multiple passband filtermanufacture.

A Rugate filter is an example of an interference coating based on therefractive index that varies continuously in a direction, for example,perpendicular or 45 degrees to the film plane. When the refractive indexvaries periodically within two extreme values, a minus filter with hightransmittance on either side of the rejection band can be made. PeriodicRugate filters can be manufactured.

Rugate notch filters can use refractory metal oxides to achieve coatingswith exceptional thermal and environmental stability. These filters canbe used in place of other types of notch filters, particularly wheredurability and reliability are desired. Rugate notch filters areavailable from Barr Associates (Westford, Mass.). The Rugate notchfilter can be used as edge filters and beam splitters. Filter sizes orshapes are not limitations for the Rugate notch filter. The Rugate notchfilter can provide environmental and thermal stability, a broadoperating temperature range, narrow rejection bands, variety of shapes &sizes, high throughput, low ripple, and/or a broad spectral range. Moreinformation is available from, for example, www.barr-associates-uk.com,www.barrassociates.com/opticalfilters.php.

Multiple-notch filters can be made, for example, with a measuredblocking of O.D. 6 or better. Notch filters with this type of deepblocking level at the light wavelength can also afford high transmissionclose to the light line.

According to various embodiments, excitation levels can increase whenmultiple dyes spaced apart spectrally are irradiated with excitationbeams. This can lead to less spectral crosstalk. The dye matrix,condition number, and/or deconvolution in a system can be improved. Theincreased excitation levels can provide higher signal levels. Highersignal levels can be seen during the utilization of dyes that emit inthe “red” spectrum. The dynamic range of the system can be improved. Thesystem can reduce the compensation for variation in the emission beamintensity for various dyes.

FIG. 9 c depicts percent light transmission plotted versus wavelength.According to various embodiments, the bandpass filter can allow lightcorresponding to the “blue,” “green,” “yellow,” “red,” and “orange”markers to pass through. The regions or zones corresponding to theexcitation light of the LEDs can be blocked. According to variousembodiments, the bandpass filter can include a single bandpass filter, aseries of bandpass filters capable of filtering the light from the LEDs,a multi-notch filter, or a Rugate filter.

FIG. 9 d depicts a plot of relative emission intensity versuswavelength, expressed in nm, for the filtered light. FIG. 9 d, ineffect, provides a breakdown, by wavelength, of the light transmittedthrough the exemplary bandpass filter. As shown in FIG. 9 d, the markersexcited by the LEDs used in the example of FIGS. 9 a through 9 d emitlight in the “blue,” “green,” “yellow,” “red,” and “orange” ranges ofwavelengths. Before focusing the light signals thus filtered onto thedetector array of a CCD, a dispersion element can be used, such asgrating 28 shown in FIG. 1. The resulting image on the CCD can besimilar to that depicted in FIGS. 2 and 3, with the addition of one ormore dark zones corresponding to the blocked excitation lightwavelengths. The existence of one or more dark zones, however, does notprevent the performance of multicomponenting to determine the intrinsicdye concentrations of each band in an electropherogram. TDI can beperformed during data collection with the above-described systemembodiments.

According to various embodiments, when irradiating multiple-channelswith multiple color LEDs, the channels can be selectively masked inorder to keep the light signals generated by the markers in each channelseparate from one another. For example and as shown in FIGS. 10 and 11,in an embodiment where three channels 68 a, 68 b, 68 c, are provided,each channel can be selectively masked with a mask 70 to create threewindows 72 a, 72 b, and 72 c, in the irradiation zone 67. The mask canbe made of a metallized surface on a glass or fused silica plate that isdisposed adjacent, for example, immediately above, the channels. Themetal can be deposited in a controlled manner, for example, byphotolithography methods, to cover the entire plate except in the areasforming the windows 72 a, 72 b, and 72 c. Alternatively, windows can becut out of an optically non-transparent plate, or a plate with windowscan otherwise be formed as known to those skilled in the art.

As depicted in FIG. 11, the array 34 of a detector can be configuredsuch that it is divided into three frames 74 a, 74 b, and 74 c,respectively, corresponding to windows 72 a, 72 b, and 72 c, allowingTDI to be effected on the charges generated by light signals detectedthrough each respective window on a window-by-window basis. The numberof wavelengths can correspond to the number of channels, or can be lessthan the number of channels. The mask 70 can be a single mask withmultiple windows, or a series of masks, each mask corresponding to oneor more channels. A device with any number of channels can be masked.According to various embodiments, each channel can pass through one ormore excitation zones. Each excitation zone can be read by acorresponding detector or emissions from each exciation zone can passthrough a corresponding detection zone.

FIG. 12 a is a schematic cut-away representation of a portion ofdetector array 34 corresponding to frame 74 a for detecting lightpassing through window 72 a. As shown in FIG. 12 a, each charge 76corresponding to an analyte band moves in the migration direction M, andthe charge packets on the detector array 34 are accumulated over anintegration time. Charges can be accumulated for TDI by shifting thecharges on the detector array in the migration direction M at apredetermined speed corresponding to the average speed of migration ofthe analyte bands, as suggested in FIG. 12 a by the shifting movementsdepicted by reference numeral 78. Data can be collected from frame 74 afrom the top of the frame to the bottom of the frame. Some of the lightcan be blocked by the mask, causing a variable collection efficiency, asdepicted by FIG. 12 b. The integration of the accumulated charges canoccur on a frame-by-frame basis as previously explained in relation toFIGS. 4, 5 a, and 5 b, the frame data being combined in a known mannerto create an electropherogram. It is further possible, according tovarious embodiments, to abut the frames, that is, to eliminate anydistance between them so as to combine the resulting images on detectorarray 34. According to various embodiments, the above-describedarrangements can allow the separation and detection of light signalsfrom multiple channels while permitting the simultaneous irradiation ofthose channels with multiple color irradiation sources. According tovarious embodiments, a single camera can be used to maintain a spatialseparation of light signals from each masked channel, or one camera perchannel can be used.

According to various embodiments, modulating optics useful for theelectrophoresis arrangements described herein are disclosed in U.S.application Ser. No. 09/564,790, the contents of which are incorporatedherein in their entirety by reference. For example, the modulatingoptics shown in FIG. 1 of U.S. patent application Ser. No. 09/564,790,using a cat's eye aperture of FIG. 24, can be useful in variouselectrophoresis arrangements as described herein.

According to various embodiments, an apparatus for detecting analytes ina sample can include means defining one or more channels therein havinga detection zone; means for separating a sample into analytes migratingalong the one or more channels, wherein the sample can include analytesand can be disposed in contact with a migration medium disposed withinthe one or more channels, wherein each analyte is detectable by thepresence of a marker; means for irradiating the detection zone withnon-coherent radiation, that can thereby excite markers responsive tothe radiation and which markers can emit light signals indicative ofcorresponding analytes; means for detecting the light signals bycollecting the light signals and producing corresponding charges; meansfor effecting a time delay integration of the charges produced by thelight signals within a detector array by accumulating within thedetector array the charges corresponding to light signals associatedwith at least one given analyte during an integration time of the atleast one given analyte moving across the detection zone; means forreading the accumulated charges; and means for separating the analytesbased on the accumulated charges that are read. Various embodiments ofthe above-described means have been substantially shown in and describedwith relation to FIGS. 1 through 12 b.

According to various embodiments, the apparatus can include a sortingmechanism to affect flow cytometry, or fluorescence-activated cellsorting, of the detected components of the sample in thechannel-defining member. The components can be cells, blood cells,nucleic acid sequences, or other biological sample components. Thesorting mechanism can be located after the detection zone. The sortingmechanism can be located a suitable distance from the detection zone toallow sufficient time for determination of the composition of acomponent of the sample based on the detected charges receivedcorresponding to the light signals associated with the at least onecomponent of the sample. Based on the identification of the component,the component can be directed into one of two or more channels. Allcomponents of a certain composition, charge, or other identifyingfactor, can be directed into the same respective channel.

The sorting mechanism can include, for example, use of an electrostaticforce, mechanical movement of respective channels, use of an acousticpulse to divert the detected component to a respective channel,manipulation of electric field gradients, vacuum, or other means asknown to those of ordinary skill in the art. Various examples of suchmethods of sorting are shown in FIGS. 13 a-c. For example, FIG. 13 adepicts a channel 12 including a detection zone 14 through whichradiation 20 passes. Emission radiation signals 22 can emit along thesame path as traveled by radiation 20, or along a separate path (notshown). Emission radiation signals 22 traveling along the same path asradiation 20 can be separated by modulating optics as described herein.Emission radiation signals 22 can be detected, for example, by detectionarray 34 of FIG. 1 and FIG. 5 a. An analyte or component 80 can travelvia the channel 12 past the detection zone 14. A diverter, for example,a charge inducer 85 can be situated at a distance past the detectionzone, sufficient for the detector to determine the composition orsalient features of the component 80. The charge inducer 85 can apply ano charge, a negative charge, or a positive charge to the component oranalyte 80. The component or analyte 80 having received a charge or nocharge from the charge inducer 85 can pass out of channel 12 and traveltowards one or more containers, for example, depicted as LEFT, WASTE,RIGHT in FIG. 13 a. The one or more containers can correspond, forexample, to a position directly below channel 12 for receipt of thenon-charged components, to the left of the channel 12 for receipt of acharged component 80, and to the right of the channel 12 for receipt ofan oppositely charged component 80. To direct the charged components 80,a magnetic field or electric field gradient can be applied across thearea through which the components fall towards the containers.Negatively charged components can gravitate towards the positivelycharged side of the field, or a positively charged plate, and positivelycharged components can gravitate towards the negative side of the fieldor the negatively charged plate, being directed into their respectivecontainers.

FIG. 13 b depicts an alternate embodiment of a sorting mechanism. Ananalyte or component 80 can travel through a channel 12 having adetection zone 14 through which radiation 20 passes, emitting emittedlight 22. Radiation 20 traveling along a direction before the detectionzone 14 can continue traveling along the same direction after thedetection zone 14 towards a detector (not shown). The detector and thedetector's associated radiation modulating optics can be disposedtransverse from the direction of the radiation 20, rather than along it.This transverse placement of the detector can improve detection ofemitted light 22. Emitted light 22 can be detected in the presence ofradiation 20. Emitted light 22 can be detected after separation fromradiation 20. After passing through the detection zone 14, the component80 can travel some distance before leaving the channel 12. The distancetraveled can be related to the time necessary to determine thecomposition or salient features of the component having passed throughthe detection zone 14. The composition of the component can cause adiverter 83 to divert the component 80 to a branch channel 88 orcontinue flowing along the channel 12. The diverter 80 can allowdetected component 80 to flow along channel 12. According to variousembodiments, one or more mechanical arms having a collection tube, acollecting container, or other means of collecting the component, can beused to move into position below channel 12 to collect the designatedcomponent 80. A device to move a collection tube, for example, a seriesof mechanical arms, can be used to collect components of variousnatures, in different collection tubes. A waste container or collectingbin can be located below channel 12 to collect any components not placedin a container of one of the mechanical arms. According to variousembodiments, the component can be removed from the stream of componentsleaving channel 12 into a desired branch channel 88 by some force, forexample, an acoustic force, a vacuum force, a pneumatic force, or someother means of moving the particle out of the particle stream and into adesignated container. According to various embodiments, a switchableelectric field or varying current flow can be applied to or across thebranch channel 88 using multiple electrodes, for example. The desiredbranch channel 88 can have a voltage potential across it, the otherbranch channels being allowed to “float” electrically. When a potentialis present at the desired branch channel the analyte can flow into theintended branch channel. A movement of the detected component 80 intothe electrically floating branch channels can be prevented. An analytepreviously moved to a branch channel 88 can be prevented from movinginto channel 12.

FIG. 13 c is another embodiment of a sorting system. The particles 80can travel in a channel 12 and pass through a detection zone 14. Channel12 can be in communication with branch channels or side channels 82, 84,and 86, branching therefrom. After passing the detection zone 14, acomponent 80 can travel down channel 12 for a distance sufficient foranalysis and recognition of the component 80 by the sorting system. Thecomposition of the component 80 can be used to determine a side channel82, 84, 86 for the component 80 to traverse. The component 80 can remainin the channel 12. Diverters 83′, 83″, and 83′″ can be activated todivert component 80 into one of branch channels 82, 84, 86. Thecomponent 80 can be maneuvered into the correct or corresponding branchchannel 82, 84, 86, or the component 80 can be allowed to continue alongchannel 12, by manipulation by electric field gradients, switchableelectric fields, vacuum forces, a stream or air, or by other means ofmovement as known to those of ordinary skill in the art. In particular,the device illustrated in FIG. 13 c can be part of a microdevice (notshown), for example, a microcard (not shown) containing channels 12, 82,84, and 86 as microcapillaries.

According to various embodiments, a sorting mechanism can be activatedupon detection of a component or analyte. The activation can cause adetection component to be removed from a flow stream containing thecomponent. After a delay time, a diverter can direct the detectioncomponent to be removed from the flow stream into a container or acollection bin. According to various embodiments, any sample notdirected to a container can be discarded as waste or can be used forsample recovery. According to various embodiments, one or more kinds ofcomponents can be present in the flow stream. One or more kinds ofcomponents can be detected. The diverter can direct variouslyidentified/detection components into one or more containers. Thediverter can employ a variety of methods to sort and/or collectcomponents. The methods of sorting the components, also known asconducting flow cytometry or cytofluorometry, can include the use ofmotive forces, for example, electrostatic forces, mechanical movement,electric field gradients, switchable electric fields, vacuums, streamsof air, or other motive forces as known to those of ordinary skill inthe art. According to various embodiments, the containers collecting thecomponents can be wells in a microdevice, for well, a microcard inmicrotiter format including one or more, for example, 24, 48, 96, 192,384, or more, wells. A sorter suitable for component sorting, forexample, cells, nucleotide acid sequences, can be obtained formDakoCytomation of Fort Collins, Colo. under the registered name MOFLOSorters. Further background on flow cytometry and cell sorting can beobtained in, for example, Hoy, Cell Sorting: Principles,http://www.uwcm.ac.uk/study/medicine/haema-tology/cytonetuk/documents/sort.pps(printed Apr. 14, 2004), which is incorporated herein its entirety byreference.

As shown with respect to FIGS. 13 a-c, multiple methods of manipulatinga component 80 for sorting the component based on its composition orbased on other features, can be used after component 80 has passeddetection zone 14. Flow cytometry can include coordination of thedetector, the processor for analyzing the data received from thedetector, and a control unit for controlling the motive force used tomanipulate the movement of the component 80. The flow cytometer caninclude a control for receiving and acting on a control signal that issent from a corresponding detector or time-delay integration system.Further description of optical flow cytometers can be found, forexample, in U.S. Pat. No. 6,549,275 which is incorporated herein in itsentirety by reference.

It is to be understood that various embodiments can be useful indetecting and imaging not only fluorescent labeled molecules, but alsoother chemical and/or biological cells or molecules, for example,proteins, viruses, and bacteria, nucleic acid sequences. Variousembodiments can be useful for the detecting and imaging of componentswhich can be electrophoretically or otherwise separated on a variety ofcarriers, for example, in capillary tubes, and across, on, in, orthrough slab gels, membranes, filter paper, Petri dishes, glasssubstrates, and the like.

According to various embodiments, the light source can be a LightEmitting Diode (LED). The LED can be, for example, an Organic LightEmitting Diode (OLED) an inorganic Light Emitted Diode, that can bepolymer-based or small-molecule-based (organic or inorganic), an edgeemitting diode (ELED), a Thin Film Electroluminescent Device (TFELD), ora Quantum dot based inorganic “organic LED.” The LED can include aphosphorescent OLED (PHOLED). As used herein, the terms “excitationsource,” “irradiation source,” and “light source” are usedinterchangeably.

According to various embodiments, excitation beams emitted from thelight source can diverge from the light source at an angle ofdivergence. The angle of divergence can be, for example, from about 5°to about 75° or more. The angle of divergence can be substantially wide,for example, greater than 45°, yet can be efficiently focused by use ofa lens, such as a focusing lens.

According to various embodiments, the light source can include one LightEmitting Diode (LED) or an array of LEDs. According to variousembodiments, each LED can be a high power LED that can emit greater thanor equal to about 1 mW of excitation energy. In various embodiments, ahigh power LED can emit at least about 5 mW of excitation energy. Invarious embodiments, the LED or array of LEDs can emit, for example, atleast about 50 mW, at least about 500 mW, or at least about 1 W or moreof excitation energy. A cooling device such as, but not limited to, aheat sink or fan, can be used with the LED. An array of high-poweredLEDs can be used that draws, for example, up to about 10 W of energy ormore, up to about 100 W of energy or more, or up to about 1000 W ormore. The total power draw can depend on the power of each LED and thenumber of LEDs in the array. The use of an LED array can result in asignificant reduction in power requirement over other light sources,such as, for example, a 75 W halogen light source or a 150 W halogenlight source. Exemplary LED array sources are available, for example,from Stocker Yale of Salem, N.H. under the trade name LED AREALIGHTS, orfrom Lumileds Lighting, LLC of San Jose, Calif. under the trade nameLUXEON, for example, a LUXEON STAR. Examples of LEDs can be found athttp://www.lumileds.com/products/family.cfm?familyId=1. According tovarious embodiments, LED light sources can use about 1 milliwatt (mW) ormore of power, for example, about 25 mW or more, about 50 mW or more,about 1 W or more, about 5 W more, about 50 W or more, or about 100 W ormore, individually or when in used in an array.

According to various embodiments, a quantum dot can be used as a sourcefor luminescence and as a fluorescent marker. Quantum dots can be usedfor both. The quantum dot based LED can be tuned to emit light in atighter emission bandpass, for example, an emission bandpass including afull-width of half-max of about 10 nm or less, about 20 nm or less, orabout 50 nm or less. The quantum dot based LED can increase theefficiency of the fluorescent system. The efficiency of a quantum dotbased LED can theoretically be higher than that of conventional LEDs,potentially over 90% when sandwiched directly between two conductivefilms with each film directly touching each quantum dot as opposed tothe present 20% efficiency for standard LEDs. Quantum dot based LEDs canbe made utilizing a slurry of quantum dots, where current flows throughan average of several quantum dots before being emitted as a photon.This conduction through several quantum dots can cause resistive lossesin efficiency. Quantum dots can provide many more colors thanconventional LEDs.

Quantum dots can be molecular-scale optical beacons. The quantum dotnanocrystals can behave like molecular LEDs (light emitting diodes) by“lighting up” biological binding events with a broad palette of appliedcolors. Quantum dots can provide many more colors than conventionalfluorophores. Quantum dots can possess many other very desirable opticalproperties. Nanocrystal quantum dots can be covalently linked tobiomolecules using standard conjugation chemistry. The quantum dotconjugate can then be used to detect a binding partner in a wide rangeof assays. According to various embodiments, streptavidin can beattached to quantum dots to detect biotinylated molecules in a varietyof assays. Quantum dots can also be attached to antibodies andoligonucleotides. Any assay that currently uses, for example,fluorescent-tagged molecules, colorimetric enzymes, or colloidal gold,can be improved with quantum dot nanocrystal-tagged conjugates. Anexemplary quantum dot implementation is available from Quantum DotCorporation of Haywood, Calif. under the trademark QDOT. Moreinformation about quantum dots and their applications can be found at,for example, www.qdots.com, and in U.S. Pat. Nos. 6,207,229, 6,251,303,6,306,310, 6,319,426, 6,322,901, 6,326,144, 6,426,513, and 6,444,143 toBawendi et al., U.S. Pat. Nos. 5,990,479, 6,207,392, and 6,423,551 toWeiss et al., U.S. Pat. No. 6,468,808 to Nie et al., and U.S. Pat. No.6,274,323 to Bruchez et al., which describe a variety of biologicalapplications, methods of quantum dot manufacturing, and apparatuses forquantum dot nanocrystals and conjugates, all of which are incorporatedherein by reference in their entireties.

Quantum dots can provide a versatile probe that can be used in, forexample, in multiplex assays. Fluorescent techniques using quantum dotnanocrystals can be much faster than conventional enzymatic andchemiluminescent techniques, can reduce instrument tie-up, and canimprove assay throughput. Colorimetric or detected reflectancetechniques can be inferior to fluorescence and difficulties ensue whenmultiplex assays are developed based on these materials. Quantum dotscan absorb all wavelengths “bluer” (i.e., shorter) than the emissionwavelength. This capability can simplify the instrumentation requiredfor multiplexed assays, since all different label colors can be excitedwith a single excitation source.

A Quantum dot based LED can emit light in an emission band that isnarrower than an emission band of a normal LED, for example, about 50%narrower or about 25% narrower. The emission band of the quantum dotscan be a function of the size distribution of the quantum dots, and thuscan theoretically be extremely narrow. The Quantum dot based LED canalso emit light at an electrical energy conversion efficiency of about,90% or more, for example, approaching 100%. OLED films, includingQuantum dot based LEDs, can be applied to a thermal block, used forheating and cooling samples, in a fluorescence system withoutinterfering with the operation of the thermal block.

According to various embodiments, when an OLED is used, the OLED canhave any of a variety of sizes, shapes, wavelengths, or combinationsthereof. The OLED can provide luminescence over a large area, forexample, to luminescence multiple sample wells. Scatter or cross-talklight between multiple sample wells for this single OLED can be reducedby either overlaying a mask on the OLED or by patterning the luminescentin the OLED to operatively align with the multiple sample wells. TheOLED can be a low power consumption device. Examples of OLEDs in variousconfigurations and wavelengths are described in, for example, U.S. Pat.No. 6,331,438 B1, which is incorporated herein by reference in itsentirety. The OLED can include a small-molecule OLED and/or apolymer-based OLED also known as a Light-Emitting Polymer (LEP). Asmall-molecule OLED that is deposited on a substrate can be used. AnOLED that is deposited on a surface by vapor-deposition technique can beused. An OLED can be deposited on a surface by, for example,silk-screening. An LEP can be used that is deposited by, for example,solvent coating.

According to various embodiments, an OLED is used and can be formed fromone or more stable, organic materials. The OLED can include one or morecarbon-based thin films and the OLED can be capable of emitting light ofvarious colors when a voltage is applied across the one or morecarbon-based thin films. Various LEDs can use different films, forexample, quantum dot based LEDs, can use Indium tin oxide.

According to various embodiments, the OLED can include a film that islocated between two electrodes. The electrodes can be, for example, atransparent anode, a metallic cathode, or combinations thereof. Severalseparate emission areas can be stimulated between a single set ofelectrodes where simultaneous illumination of the separate emissionareas is required. According to such embodiments, only one power andcontrol module might be required for several apparent light sources. TheOLED film can include one or more of a hole-injection layer, ahole-transport layer, an emissive layer, and an electron-transportlayer. The OLED can include a film that is about one micrometer inthickness, or less. When an appropriate voltage is applied to the film,the injected positive and negative charges can recombine in the emissivelayer to produce light by means of electroluminescence. The amount oflight emitted by the OLED can be related to the voltage applied throughthe electrodes to the thin film of the OLED. Various materials suitablefor fabrication of OLEDs are available, for example, from H.W. SandsCorp. of Jupiter, Fla. Various types of OLEDs are described, forexample, in U.S. Pat. No. 4,356,429 to Tang, U.S. Pat. No. 5,554,450 toShi et al., and U.S. Pat. No. 5,593,788 to Shi et al., all of which areincorporated herein in their entireties by reference.

According to various embodiments, an OLED can be used and/or produced ona flexible substrate, on an optically clear substrate, on a substrate ofan unusual shape, or on a combination thereof. Multiple OLEDs can becombined on a substrate, wherein the multiple OLEDs can emit light atdifferent wavelengths. Multiple OLEDs on a single substrate or multipleadjacent substrates can form an interlaced or a non-interlaced patternof light of various wavelengths. The pattern can correspond to, forexample, a sample reservoir arrangement. One or more OLEDs can form ashape surrounding, for example, a sample reservoir, a series of samplereservoirs, an array of a plurality of sample reservoirs, or a sampleflow path. The sample path can be, for example, a channel, a capillary,or a micro-capillary. One or more OLEDs can be formed to follow thesample flow path. One or more OLEDs can be formed in the shape of asubstrate or a portion of a substrate. For example, the OLED can becurved, circular, oval, rectangular, square, triangular, annular, or anyother geometrically regular shape. The OLED can be formed as anirregular geometric shape. The OLED can illuminate one or more samplereservoirs, for example, an OLED can illuminate one, two, three, four,or more sample reservoirs simultaneously, or in sequence. The OLED canbe designed, for example, to illuminate all the wells of a correspondingmulti-well array.

According to various embodiments, one or more excitation filters can beincorporated into the OLED substrate, thus eliminating additionalequipment and reducing the amount of space needed for an optical system.For example, one or more filters can be formed in a layer of a substrateincluding one or more OLEDs and a layer including a sample flow path.The wavelength emitted by the OLED can be tuned by printing afluorescent dye in the OLED substrate, as taught, for example, by Hebneret al. in “Local Tuning of Organic Light-Emitting Diode Color by DyeDroplet Application,” APPLIED PHYSICS LETTERS, Vol. 73, No. 13 (Sep. 28,1998), which is incorporated herein by reference in its entirety. Whenusing multiple emission lines in an OLED, the OLED can be used incombination with a multiple notch emission filter.

According to various embodiments, an OLED can be substituted for an LEDin any of the systems, devices, apparatuses, and methods describedherein wherein an LED is included or used. The OLED light source canhave several OLED films stacked and operatively disposed such thatseveral wavelengths of excitation beams can traverse the same opticalpath to illuminate the sample well. Several OLEDs forming excitationbeams of the same wavelength can be stacked to provide higher output toilluminate the sample well.

According to various embodiments, an ELED can be substituted for an LEDin any of the systems, devices, apparatuses, and methods describedherein wherein an LED is included or used. The ELED light source can bea light-emitting diode with output that emanates from betweenheterogeneous layers. An ELED can have greater radiance and couplingefficiency to an optical fiber or integrated optical circuit than asurface-emitting LED.

According to various embodiments, the light source can be a Solid StateLaser (SSL) or a micro-wire laser. The SSL can produce monochromatic,coherent, directional light and can provide a narrow wavelength ofexcitation energy. The SSL can use a lasing material that is distributedin a solid matrix, in contrast to other lasers that use a gas, dye, orsemiconductor for the lasing source material. Examples of solid statelasing materials and corresponding emission wavelengths can include, forexample: Ruby at about 694 nm; Nd:Yag at about 1064 nm; Nd:YVO4 at about1064 nm and/or about 1340 nm and which can be doubled to emit at about532 nm or about 670 nm; Alexandrite at from about 655 nm to about 815nm; and Ti:Sapphire at from about 840 nm to about 1100 nm. Micro-wirelasers are lasers where the wavelength of an excitation beam formed bythe laser can be tuned or adjusted by altering the size of a wire.According to various embodiments, other solid state lasers known tothose skilled in the art can also be used, for example, laser diodes.The appropriate lasing material can be selected based on the fluorescingdyes used, the excitation wavelength required, or both.

According to various embodiments, a Vertical Cavity Laser (VCL) can beused as an excitation source. A VCL can be substituted for an LED in anyof the devices, systems, apparatuses, or methods described hereinwherein an LED is included or used. A VCL can be a type ofsurface-emitting laser diode that uses dielectric mirrors to producesurface emission. The laser cavity can be established in a verticaldirection with respect to the plane of the active region. Examples,uses, and descriptions of VCL's can be found in U.S. Pat. No. 4,999,842,for example, that is incorporated herein in its entirety by reference.

If a SSL is used, the laser can be selected to closely match theexcitation wavelength of a fluorescent dye. The operating temperature ofthe system can be considered in selecting an appropriate SSL. Theoperating temperature can be regulated or controlled to change theemitted wavelength of the SSL. The light source for the laser can be anysource as known to those skilled in the art, such as, for example, aflash lamp. Useful information about various solid state lasers can befound at, for example, www.repairfaq.org/sam/lasersl.htm. Examples ofsolid state lasers used in various systems for identification ofbiological materials can be found in, for example, U.S. Pat. No.5,863,502 to Southgate et al. and U.S. Pat. No. 6,529,275 B2 toAmirkhanian et al.; both of which are incorporated herein by referencein their entireties.

According to various embodiments, various types of light sources can beused singularly or in combination with other light sources. One or moreOLEDs can be used with, for example, one or more non-organic LEDs, oneor more solid state lasers, one or more halogen light sources, orcombinations thereof.

According to various embodiments, an OLED layout can be connected to thepower supply through leads arranged at opposite corners of the OLEDlayout. The power supply can include or be connected to one or more of aswitch, a meter, an oscillator, a potentiometer, a detector, a signalprocessing unit, or the like. The OLED layout can include a plurality ofindividually addressable OLED lighting elements (not shown) with aseparate lead connected to each lighting element. The wiring, leads,terminals, connection arms, and the like can be implemented in, forexample, a substrate or a film. The OLED layout can be shaped to bealigned with, for example, a plurality of detection zones. Otherembodiments of OLED layouts using various shapes and various numbers ofwell lamps are within the scope of the present teachings.

According to various embodiments, each individually addressable OLEDlighting elements can include, for example, four individual lamps orOLED layers, capable of producing excitation wavelengths at fourdifferent frequencies.

The OLED layout can be constructed of a unitary or multi-partconstruction, of molded material, of stamped material, of screen printedmaterial, of cut material, or the like.

FIG. 14 illustrates an exemplary embodiment of a light source layout. AnOLED layout 450 can include varying color OLEDs 452, 454, and 456stacked upon each other. The layout can be useful for a compact lightsource design capable of forming excitation beams at varyingwavelengths. The OLEDs 452, 454, and 456 can be transparent, allowingexcitation beams from each OLED to pass through any other OLED so as tobe directed towards a sample. The OLEDs 452, 454, and 456 can emitdifferent colors, same colors, or a combination thereof depending on thecolor intensity and variety required. The OLEDs 452, 454, and 456 canshare an electrode, for example, a cathode. One electrode, for example,an anode, for powering each of the OLEDs 452, 454, and 456 can beconnected in electrical isolation from each respective anode to acontrol unit (not shown) if the capability to independently activateeach of the OLEDs 452, 454, and 456 is desired. The OLEDs 452, 454, and456 can electrically share one electrode, two electrodes, or noelectrodes. Any number of OLEDs can be stacked, for example, two OLEDs,three OLEDs, four OLEDs, or more OLEDs, to form a light source, arespective light source, or an array of light sources.

According to various embodiments, multiple excitation wavelengths can beused to detect multiple sample components. According to variousembodiments, the apparatus and method can be adapted for use by anysuitable fluorescence detection system. For example, various embodimentsof the apparatus and method can be used in a sequencing system withsingle or multiple samples, for example, in a nucleic acid sequenceamplification reaction, in a sequencing detection system.

Various embodiments of the teachings are described herein. The teachingsare not limited to the specific embodiments described, but encompassequivalent features and methods as known to one of ordinary skill in theart. Other embodiments will be apparent to those skilled in the art fromconsideration of the present specification and practice of the teachingsdisclosed herein. It is intended that the present specification andexamples be considered as exemplary only.

1. An apparatus for detecting analytes in a sample containing at leastone analyte, the apparatus comprising: one or more channels having adetection zone; one or more irradiation sources disposed for irradiatingthe detection zone with radiation; a detector capable of collecting atleast one charge corresponding to an emission beam emitted from thedetection zone, the detector having an output; a time delay integrationsystem coupled to the detector for effecting time delay integration ofthe at least one charge by accumulating the at least one charge beforereading the at least one charge at the output of the detector, andmodulating optics including a multi-notch filter disposed along a beampath between at least one of the one or more irradiation sources and thedetection zone, and the detection zone and the detector.
 2. Theapparatus of claim 1, wherein the time delay integration systemaccumulates the at least one charge by moving, relative to one another,the modulating optics and the one or more channels.
 3. The apparatus ofclaim 1, wherein the one or more irradiation sources comprises one ormore light emitting diodes.
 4. The apparatus of claim 3, wherein the oneor more light emitting diodes compnses one or more organic lightemitting diodes.
 5. The apparatus of claim 3, wherein the one or morelight emitting diodes comprises one or more quantum dots.
 6. Theapparatus of claim 1, wherein the one or more irradiation sourcescomprises one or more lasers.
 7. The apparatus of claim 6, wherein theone or more lasers comprises one or more solid state lasers.
 8. Theapparatus of claim 6, wherein the one or more lasers comprises one ormore micro-wire lasers.
 9. The apparatus of claim 1, wherein themodulating optics includes a relay lens system providing a collimatinglens and a re-imaging lens.
 10. The apparatus of claim 1, wherein themodulating optics includes a focusing lens disposed between themulti-notch filter and the detection zone.
 11. The apparatus of claim 1,further comprising a separating system coupled to the one or morechannels for separating the sample containing the at least one analyteinto at least one analyte band migrating along the one or more channelsupon detection of the emission beam.
 12. The apparatus of claim 1,wherein the at least one analyte is labeled with at least one marker.13. The apparatus of claim 12, wherein the at least one marker comprisesa dye marker, a fluorescing dye, a free-floating dye, a reporter dye, aprobe dye, an intercalating dye, a quantum dot, a molecular beacon, alinear probe, or a combination thereof.
 14. The apparatus of claim 12,wherein the multi-notch filter provides at least one conditioning filterfor the one or more irradiation sources, each conditioning filter beingeffective for substantially blocking the radiation at predeterminedexcitation wavelengths to produce conditioned light, wherein thepredetermined excitation wavelengths of each marker do not conflict withthe excitation spectra of each other marker.
 15. The apparatus of claim14, wherein the modulating optics comprises a second multi-notch filterincluding at least one conditioning filter for at least one emissionspectra of the at least one marker, each respective conditioning filterbeing effective for substantially blocking predetermined emissionwavelengths of at least one emission spectra of each marker to produceconditioned light, wherein the predetermined emission wavelengths ofeach marker do not conflict with the emission spectra of each othermarker.
 16. The apparatus of claim 14, further comprising an offsetsystem for spatially offsetting on fine detector at least one image foreach conditioning filter by a predetermined amount.
 17. The apparatus ofclaim 16, wherein the offset system comprises a plurality of offsetmechanisms, each associated with a respective one of the conditioningfilters.
 18. The apparatus of claim 17, wherein each offset mechanismincludes one or more of a glass plate, a grating, a minor, or acombination thereof.
 19. The apparatus of claim 16, wherein the offsetsystem is adapted to effect a translational movement of at least one ofthe detector, the modulating optics, and the detection zone, withrespect to one another, for spatially offsetting the at least one imageby a second predetermined amount.
 20. The apparatus of claim 14, whereinthe time delay integration system is capable of controlling the detectorto read the at least one accumulated charge on a frame by frame basis,each frame corresponding to the at least one accumulated charge on thedetector during an integration time and produced by the conditionedlight through each conditioning filter.
 21. The apparatus of claim 1,wherein the time delay integration system is capable of controlling thedetector to read the at least one accumulated charge on a continuousbasis.
 22. The apparatus of claim 1, wherein the detector comprises atwo-dimensional charge-coupled device.
 23. The apparatus of claim 11,wherein the time delay integration system includes a system coupled tothe modulating optics for moving the modulating optics relative to theone or more channels at a speed that is synchronized to a migration ofthe at least one analyte band across the detection zone.
 24. Theapparatus of claim 11, wherein the time delay integration systemcomprises a system coupled to the one or more channels for moving theone or more channels relative to the modulating optics at a speed thatis synchronized to a migration of the at least one analyte band acrossthe detection zone.
 25. The apparatus of claim 11, further including asorting system including one or more collection channels and acontroller for controlling a motive force for each collection channel,wherein the sorting system is capable of sorting each analyte into therespective collection channel.
 26. The apparatus of claim 25, whereinthe motive force comprises an electrokinetic force, a mechanical force,a production of electric field gradient, a switchable electric field,vacuum, or a combination thereof.
 27. The apparatus of claim 25, whereinthe controller comprises one or more controllers each associated with arespective collection channel.
 28. The apparatus of claim 1, wherein theone or more irradiation sources comprises a mechanism for spectrallydistributing the radiation thereby producing spectrally distributedradiation.
 29. The apparatus of claim 1, wherein the multi-notch filteris a Rugate filter.
 30. An apparatus for detecting analytes in a samplecontaining at least one analyte, the apparatus comprising: one or morechannels having a detection zone: one or more irradiation sourcesdisposed for irradiating the detection zone with radiation, wherein theone or more irradiation sources comprises one or more of a solid statelaser, a micro-wire laser, an organic light-emitting diode, an edgeemitting diode, a vertical cavity laser, and a quantum-dot-based LED; adetector capable of collecting at least one charge corresponding to anemission beam emitted from the detection zone, the detector having anoutput; modulating optics along a beam path between the one or moreirradiation sources and the detector; and a time delay integrationsystem coupled to the detector for effecting time delay integration ofthe at least one charge by accumulating the at least one charge beforereading the at least one charge at the output of the detector, whereinthe time delay integration system accumulates the at least one charge bymoving, relative to one another, the modulating optics and the one ormore channels.
 31. An apparatus for detecting analytes in a samplecontaining at least one analyte, the apparatus comprising: one or morechannels having a detection zone; one or more irradiation sourcesdisposed for irradiating the detection zone with radiation, wherein theone or more irradiation sources comprises one or more of a solid statelaser, a micro-wire laser, an organic light-emitting diode, an edgeemitting diode, a vertical cavity laser, and a quantum-dot-based LED; adetector capable of collecting at least one charge corresponding to anemission beam emitted from the detection zone, the detector having anoutput; modulating optics along a beam path between the one or moreirradiation sources and the detector, wherein the modulating opticsincludes a relay lens system providing a collimating lens and are-imaging lens; and a time delay integration system coupled to thedetector for effecting time delay integration of the at least one chargeby accumulating the at least one charge before reading the at least onecharge at the output of the detector.
 32. An apparatus for detectinganalytes in a sample containing at least one analyte, the apparatuscomprising: one or more channels having a detection zone; one or moreirradiation sources disposed for irradiating the detection zone withradiation, wherein the one or more irradiation sources comprises one ormore of a solid state laser, a micro-wire laser, an organiclight-emitting diode, an edge emitting diode, a vertical cavity laser,and a quantum-dot-based LED; a detector capable of collecting at leastone charge corresponding to an emission beam emitted from the detectionzone, the detector having an output; modulating optics along a beam pathbetween the one or more irradiation sources and the detector, whereinthe modulating optics includes a focusing lens disposed between amulti-notch filter and the detection zone; and a time delay integrationsystem coupled to the detector for effecting time delay integration ofthe at least one charge by accumulating the at least one charge beforereading the at least one charge at the output of the detector.
 33. Anapparatus for detecting analytes in a sample containing at least oneanalyte, the apparatus comprising: one or more channels having adetection zone; one or more irradiation sources disposed for irradiatingthe detection zone with radiation, wherein the one or more irradiationsources comprises one or more of a solid state laser, a micro-wirelaser, an organic light-emitting diode, an edge emitting diode, avertical cavity laser, and a quantum-dot-based LED; a detector capableof collecting at least one charge corresponding to an emission beamemitted from the detection zone, the detector having an output;modulating optics along a beam path between the one or more irradiationsources and the detector, the modulating optics providing at least oneconditioning filter for the one or more irradiation sources, eachconditioning filter being effective for substantially blocking theradiation at predetermined excitation wavelengths to produce conditionedlight, wherein the predetermined excitation wavelengths of each markerdo not conflict with the excitation spectra of each other marker, themodulating optics comprising a second filter set including at least oneconditioning filter for at least one emission spectra of the at leastone marker, each respective conditioning filter being effective forsubstantially blocking predetermined emission wavelengths of at leastone emission spectra of each marker to produce conditioned light,wherein the predetermined emission wavelengths of each marker do notconflict with the emission spectra of each other marker; and a timedelay integration system coupled to the detector for effecting timedelay integration of the at least one charge by accumulating the atleast one charge before reading the at least one charge at the output ofthe detector.
 34. The apparatus of claim 33, further comprising anoffset system for spatially offsetting on the detector at least oneimage for each conditioning filter by a predetermined amount.
 35. Theapparatus of claim 34, wherein the offset system comprises a pluralityof offset mechanisms, each associated with a respective one of theconditioning filters.
 36. The apparatus of claim 35, wherein each offsetmechanism includes one or more of a glass plate, a grating, a mirror, ora combination thereof.
 37. The apparatus of claim 34, wherein the offsetsystem is adapted to effect a translational movement of at least one ofthe detector, a modulating optics along a beam path between the one ormore irradiation sources and the detector, and the detection zone, withrespect to one another, for spatially offsetting the at least one imageby a second predetermined amount.
 38. An apparatus for detectinganalytes in a sample containing at least one analyte, the apparatuscomprising: one or more channels having a detection zone; one or moreirradiation sources disposed for irradiating the detection zone withradiation, wherein the one or more irradiation sources comprises one ormore of a solid state laser, a micro-wire laser, an organiclight-emitting diode, an edge emitting diode, a vertical cavity laser,and a quantum-dot-based LED; a detector capable of collecting at leastone charge corresponding to an emission beam emitted from the detectionzone, the detector having an output; modulating optics along a beam pathbetween the one or more irradiation sources and the detector, themodulating optics providing at least one conditioning filter for the oneor more irradiation sources, each conditioning filter being effectivefor substantially blocking the radiation at predetermined excitationwavelengths to produce conditioned light, wherein the predeterminedexcitation wavelengths of each marker do not conflict with theexcitation spectra of each other marker; and a time delay integrationsystem coupled to the detector for effecting time delay integration ofthe at least one charge by accumulating the at least one charge beforereading the at least one charge at the output of the detector, whereinthe time delay integration system is capable of controlling the detectorto read the at least one accumulated charge on a frame by frame basis,each frame corresponding to the at least one accumulated charge on thedetector during an integration time and produced by the conditionedlight through each conditioning filter.
 39. An apparatus for detectinganalytes in a sample containing at least one analyte, the apparatuscomprising: one or more channels having a detection zone: one or moreirradiation sources disposed for irradiating the detection zone withradiation, wherein the one or more irradiation sources comprises one ormore of a solid state laser, a micro-wire laser, an organiclight-emitting diode, an edge emitting diode, a vertical cavity laser,and a quantum-dot-based LED; a detector capable of collecting at leastone charge corresponding to an emission beam emitted from the detectionzone, the detector having an output; a separating system coupled to theone or more channels for separating the sample containing the at leastone analyte into at least one analyte band migrating along the one ormore channels upon detection of the emission beam; and a time delayintegration system coupled to the detector for effecting time delayintegration of the at least one charge by accumulating the at least onecharge before reading the at least one charge at the output of thedetector, wherein the time delay integration system includes a systemcoupled to the modulating optics for moving the modulating opticsrelative to the one or more channels at a speed that is synchronized toa migration of the at least one analyte band across the detection zone.40. An apparatus for detecting analytes in a sample containing at leastone analyte, the apparatus comprising: one or more channels having adetection zone; one or more irradiation sources disposed for irradiatingthe detection zone with radiation, wherein the one or more irradiationsources comprises one or more of a solid state laser, a micro-wirelaser, an organic light-emitting diode, an edge emitting diode, avertical cavity laser, and a quantum-dot-based LED; a detector capableof collecting at least one charge corresponding to an emission beamemitted from the detection zone, the detector having an output; aseparating system coupled to the one or more channels for separating thesample containing the at least one analyte into at least one analyteband migrating along the one or more channels upon detection of theemission beam; and a time delay integration system coupled to thedetector for effecting time delay integration of the at least one chargeby accumulating the at least one charge before reading the at least onecharge at the output of the detector, wherein the time delay integrationsystem comprises a system coupled to the one or more channels for movingthe one or more channels relative to the modulating optics at a speedthat is synchronized to a migration of the analyte bands across thedetection zone.
 41. An apparatus for detecting analytes in a samplecontaining at least one analyte the apparatus comprising: one or morechannels having a detection zone; one or more irradiation sourcesdisposed for irradiating the detection zone with radiation, wherein theone or more irradiation sources comprises one or more of a solid statelaser, a micro-wire laser, an organic light-emitting diode, an edgeemitting diode, a vertical cavity laser, and a quantum-dot-based LED; adetector capable of collecting at least one charge corresponding to anemission beam emitted from the detection zone, the detector having anoutput; modulating optics including a multi-notch filter disposed alonga beam path between at least one of the one or more irradiation sourcesand the detection zone, and the detection zone and the detector, whereinthe multi-notch filter is a Rugate; and a time delay integration systemcoupled to the detector for effecting time delay integration of the atleast one charge by accumulating the at least one charge before readingthe at least one charge at the output of the detector.
 42. A method fordetecting analytes in a sample containing at least one analyte and atleast one marker, comprising the steps of: providing one or morechannels having a detection zone; providing migration medium within theone or more channels; separating a sample containing analytes anddisposed in contact with the migration medium into at least one analyteband migrating along the one or more channels, wherein each analyte bandis detectable by the presence of the at least one marker; irradiatingthe detection zone using one or more irradiation sources disposed forirradiating the detection zone with radiation so that the at least onemarker is responsive to the radiation to produce emission beamsindicative of corresponding analytes; detecting the emission beams on adetector to produce at least one charge on the detector corresponding tothe emission beams; modulating light between the one or more irradiationsources and the detector wherein modulating the light comprisesdirecting the radiation through a multi-notch filter; effecting a timedelay integration of the at least one charge by accumulating the atleast one charge associated with at least one given analyte band duringan integration time of the at least one given analyte before reading theat least one charge at the output of the detector; and reading theaccumulated charges.
 43. The method of claim 42, further comprisingseparating the sample containing at least one analyte and disposed incontact with the migration medium into at least one analyte bandmigrating along the one or more channels, wherein each analyte band isdetectable by the presence of the at least one marker.
 44. The method ofclaim 42, further comprising moving of the light signals within thedetector wherein the accumulation is effected by moving, relative to oneanother, the detector and the one or more channels.
 45. The methodaccording to claim 42, wherein the step of accumulating the at least onecharge with the detector includes at least one of the steps of shiftingthe at least one charge on the detector and moving, relative to oneanother, the detector and the emission beams from the at least onemarker.
 46. The method of claim 42, further comprising the step ofcontacting the sample with the migration medium.
 47. The method of claim42, wherein the modulating comprises: substantially blockingpredetermined wavelengths of light emitted by a respective irradiationsource of the one or more irradiation sources to thereby produceconditioned light, the predetermined wavelengths being those wavelengthsnot overlapping the emission spectra of the at least one markerresponsive to the respective one of the one or more irradiation sources;focusing the conditioned light onto the detection zone; collimatinglight from the detection zone to thereby generate collimated light; andfiltering the light from the detection zone by transmitting,substantially exclusively, at least one predetermined wavelength rangeof the light signals from the detection zone corresponding to a portionof the wavelengths of the light signals emitted by the at least onemarker, to thereby produce filtered light.
 48. The method of claim 47,further comprising the step of focusing the filtered light onto thedetector.
 49. The method of claim 42, wherein the modulating opticscomprise at least one conditioning filter for the one or moreirradiation sources, each conditioning filter being effective forsubstantially blocking the radiation at predetermined excitationwavelength to produce conditioned light, wherein the predeterminedexcitation wavelengths of each marker do not conflict with theexcitation spectra of each other marker.
 50. The method of claim 49,further comprising the step of spatially offsetting the filtered lightfrom the at least one conditioning filter by a predetermined amount as afunction of said at least one conditioning filter such that a pluralityof images on the detector is produced by the at least one charge beingspatially offset from another charge.
 51. The method of claim 42,wherein the one or more irradiation sources comprises at least one of alight emitting diode, a organic light emitting diode, a quantum dot, asolid state laser, a micro-wire laser, an edge emitting diode, avertical cavity laser, and a quantum-dot-based LED.
 52. The method ofclaim 42, further comprising sorting analytes of the sample based on thereading of the accumulating charges.
 53. The method of claim 52, whereinsorting the sample comprises administering a motive force to one or moreanalytes of the sample to control movement of the one or more analytes.54. The method of claim 52, wherein the motive force is anelectrokinetic force, mechanical force, an electrical field gradient, aswitchable electric field, a vacuum, or an air stream.
 55. A method fordetecting analytes in a sample containing at least one analyte and atleast one marker, comprising the steps of: providing one or morechannels having a detection zone; providing migration medium within theone or more channels; irradiating the detection zone using one or moreirradiation sources disposed for irradiating the detection zone withradiation so that the at least one marker is responsive to the radiationto produce emission beams indicative of corresponding analytes whereinthe one or more irradiation sources comprises one or more of a solidstate laser, a micro-wire laser, an organic light-emitting diode, anedge emitting diode, a vertical cavity laser, and a quantum-dot-basedLED; detecting the emission beams on a detector to produce at least onecharge on the detector corresponding to the emission beams; modulatinglight between the one or more irradiation sources and the detector;effecting a time delay integration of the at least one charge byaccumulating the at least one charge associated with at least one givenanalyte band during an integration time of the at least one givenanalyte before reading the at least one charge at the output of thedetector; moving of the light signals within the detector wherein theaccumulation is effected by moving, relative to one another, thedetector and the one or more channels; and reading the accumulatedcharges.
 56. A method for detecting analytes in a sample containing atleast one analyte and at least one marker, comprising the steps of:providing one or more channels having a detection zone; providingmigration medium within the one or more channels; irradiating thedetection zone using one or more irradiation sources disposed forirradiating the detection zone with radiation so that the at least onemarker is responsive to the radiation to produce emission beamsindicative of corresponding analytes wherein the one or more irradiationsources comprises one or more of a solid state laser, a micro-wirelaser, an organic light-emitting diode, an edge emitting diode, avertical cavity laser, and a quantum-dot-based LED; detecting theemission beams on a detector to produce at least one charge on thedetector corresponding to the emission beams; modulating light betweenthe one or more irradiation sources and the detector; effecting a timedelay integration of the at least one charge by accumulating the atleast one charge associated with at least one given analyte band duringan integration time of the at least one given analyte before reading theat least one charge at the output of the detector, the accumulating theat least one charge with the detector including at least one of shiftingthe at least one charge on the detector and moving, relative to oneanother, the detector and light signals from the detection zone; andreading the accumulated charges.
 57. A method for detecting analytes ina sample containing at least one analyte and at least one marker,comprising the steps of: providing one or more channels having adetection zone; providing migration medium within the one or morechannels; irradiating the detection zone using one or more irradiationsources disposed for irradiating the detection zone with radiation sothat the at least one marker is responsive to the radiation to produceemission beams indicative of corresponding analytes wherein the one ormore irradiation sources comprises one or more of a solid state laser, amicro-wire laser, an organic light-emitting diode, an edge emittingdiode, a vertical cavity laser, and a quantum-dot-based LED; detectingthe emission beams on a detector to produce at least one charge on thedetector corresponding to the emission beams; modulating light betweenthe one or more irradiation sources and the detector, wherein themodulating comprises; substantially blocking predetermined wavelengthsof light emitted by a respective irradiation source of the one or moreirradiation sources to thereby produce conditioned light, thepredetermined wavelengths being those wavelengths not overlapping theemission spectra of the at least one marker responsive to the respectiveone of the one or more irradiation sources; focusing the conditionedlight onto the detection zone; collimating light from the detection zoneto thereby generate collimated light; and filtering the light from thedetection zone by transmitting, substantially exclusively, at least onepredetermined wavelength range of the light signals from the detectionzone corresponding to a portion of the wavelengths of the light signalsemitted by the at least one marker, to thereby produce filtered light;effecting a time delay integration of the at least one charge byaccumulating the at least one charge associated with at least one givenanalyte band during an integration time of the at least one givenanalyte before reading the at least one charge at the output of thedetector; and reading the accumulated charges.
 58. The method of claim57, further comprising the step of focusing the filtered light onto thedetector.
 59. A method for detecting analytes in a sample containing atleast one analyte and at least one marker, comprising the steps of:providing one or more channels having a detection zone; providingmigration medium within the one or more channels; contacting the samplewith the migration medium; irradiating the detection zone using one ormore irradiation sources disposed for irradiating the detection zonewith radiation so that the at least one marker is responsive to theradiation to produce emission beams indicative of corresponding analyteswherein the one or more irradiation sources comprises one or more of asolid state laser, a micro-wire laser, an organic light-emitting diode,an edge emitting diode, a vertical cavity laser, and a quantum-dot-basedLED; detecting the emission beams on a detector to produce at least onecharge on the detector corresponding to the emission beams; modulatinglight between the one or more irradiation sources and the detector, themodulating the light comprising directing the radiation throughmodulating optics comprising a multi-notch filter, the multi-notchfilter providing at least one conditioning filter for the one or moreirradiation sources, each conditioning filter being effective forsubstantially blocking the radiation at predetermined excitationwavelengths to produce conditioned light, wherein the predeterminedexcitation wavelengths of each marker do not conflict with theexcitation spectra of each other marker; effecting a time delayintegration of the at least one charge by accumulating the at least onecharge associated with at least one given analyte band during anintegration time of the at least one given analyte before reading the atleast one charge at the output of the detector; and reading theaccumulated charges.
 60. The method of claim 59, wherein the modulatingoptics comprises a second multi-notch filter including at least oneconditioning filter for at least one emission spectra of the at leastone marker, each respective conditioning filter being effective forsubstantially blocking predetermined emission wavelengths of at leastone emission spectra of each marker to produce conditioned light,wherein the predetermined emission wavelengths of each marker do notconflict with the emission spectra of each other marker.
 61. The methodof claim 60, further comprising the step of spatially offsetting thefiltered light from each conditioning filter by a predetermined amountas a function of said each filter such that an image on the detector isproduced by the at least one charge spatially offset from on another.